Immediate release pharmaceutical formulation

ABSTRACT

According to the present invention there is provided an immediate release pharmaceutical formulation comprising, as active ingredient, a compound of formula (I):  
                 
wherein 
     R 1  represents C 1-2  alkyl substituted by one or more fluoro substituents;    R 2  represents hydrogen, hydroxy, methoxy or ethoxy; and n represents 0, 1 or 2; or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable diluent or carrier; provided that when the active ingredient is other than in the form of a salt the formulation does not solely contain: a solution of one active ingredient and water, a solution of one active ingredient and dimethylsulphoxide; or a solution of one active ingredient in a mixture of ethanol:PEG 660 12-hydroxy stearate:water 5:5:90; such formulations being of use for the treatment of a cardiovascular disorder.

This application is a national stage filing under 35 U.S.C. 371 ofInternational Application No. PCT/SE03/00857, filed May 27, 2003, whichclaims priority from Sweeden Application No. 0201658-2, filed May 31,2002, the specification of which is incorporated by reference herein.International Application No. PCT/SE03/00857 was published under PCTArticle 21(2) in English.

This invention relates to a novel immediate release pharmaceuticalformulation that provides for the delivery of particularpharmaceuticals, to the manufacture of such a formulation, and to theuse of such a formulation in the treatment or prevention of thrombosis.

It is often desirable to formulate pharmaceutically active compounds forimmediate release following oral and/or parenteral administration with aview to providing a sufficient concentration of drug in plasma withinthe time-frame required to give rise to a desired therapeutic response.

Immediate release may be particularly desirable in cases where, forexample, a rapid therapeutic response is required (for example in thetreatment of acute problems), or, in the case of parenteraladministration, when peroral delivery to the gastrointestinal tract isincapable of providing sufficient systemic uptake within the requiredtime-frame.

In the case of the treatment or prophylaxis of thrombosis, immediaterelease formulations may be necessary to ensure that a sufficient amountof drug is provided in plasma within a relatively short period of timeto enable quick onset of action. Immediate release formulations are alsotypically simpler to develop than modified release formulations, and mayalso provide more flexibility in relation to the variation of doses thatare to be administered to patients. Immediate release formulations aresuperior when multiple doses are not required and where it is notnecessary to keep the plasma concentration at a constant level for anextended time.

International Patent Application No. PCT/SE01/02657 (WO 02/44145,earliest priority date 1 Dec. 2000, filed 30 Nov. 2001, published 6 Jun.2002) discloses a number of compounds that are, or are metabolised tocompounds which are, competitive inhibitors of trypsin-like proteases,such as thrombin. The following three compounds are amongst those thatare specifically disclosed:

-   (a) Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(OMe):    which compound is referred to hereinafter as Compound A;-   (b) Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(OMe):    which compound is referred to hereinafter as Compound B; and-   (c) Ph(3-Cl)(5-OCH₂CH₂F)—(R)CH(OH)C(O)—(S)Aze-Pab(OMe):    which compound is referred to hereinafter as Compound C.

The methoxyamidine Compounds A, B and C are metabolised following oraland/or parenteral administration to the corresponding free amidinecompounds, which latter compounds have been found to be potentinhibitors of thrombin. Thus:

-   -   Compound A is metabolized to        Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab (which compound is        referred to hereinafter as Compound D) via a prodrug        intermediate Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(OH)        (which compound is referred to hereinafter as Compound G);    -   Compound B is metabolized to        Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF) (which        compound is referred to hereinafter as Compound E) via a prodrug        intermediate        Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(OH) (which        compound is referred to hereinafter as Compound H); and,    -   Compound C is metabolized to        Ph(3-Cl)(5-OCH₂CH₂F)—(R)CH(OH)C(O)—(S)Aze-Pab (which compound is        referred to hereinafter as Compound F) via a prodrug        intermediate Ph(3-Cl)(5-OCH₂CH₂F)—(R)CH(OH)C(O)—(S)Aze-Pab(OH)        (which compound is referred to hereinafter as Compound J).

Processes for the synthesis of Compounds A, B, C, D, E, F, G and J aredescribed in Examples 12, 40, 22, 3, 39, 21, 2 and 31 (respectively) ofinternational patent application No. PCT/SE01/02657. An immediaterelease formulation of these compounds, or their metabolites has yet tobe described in the literature. We have found that the compounds offormula (I) and their salts can be formulated as immediate releasepharmaceutical formulations which are easy to administer, for example byoral or parenteral administration.

According to a first aspect of the invention, there is provided animmediate release pharmaceutical formulation comprising, as activeingredient, a compound of formula (I):

wherein

-   R¹ represents C₁₋₂ alkyl substituted by one or more fluoro    substituents;-   R² represents hydrogen, hydroxy, methoxy or ethoxy; and-   n represents 0, 1 or 2;    or a pharmaceutically acceptable salt thereof; and a    pharmaceutically acceptable diluent or carrier; provided that the    formulation does not solely contain:    -   a solution of one active ingredient and water;    -   a solution of one active ingredient and dimethylsulphoxide; or,    -   a solution of one active ingredient in a mixture of ethanol:PEG        660 12-hydroxy stearate:water 5:5:90;        which formulations are referred to hereinafter as “the        formulations of the invention”.

PEG 660 12-hydroxy stearate is a non-ionic surfactant and is betterknown as Solutol K™.

According to a second aspect of the present invention there is providedCompound H, Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(OH)which can be prepared by methods similar to those described below forthe preparation of Compounds G and J.

The compounds of formula (I), or a pharmaceutically acceptable saltthereof, may be in the form of a solvate, a hydrate, a mixedsolvate/hydrate or, preferably, an ansolvate, such as an anhydrate.Solvates may be of one or more organic solvents, such as lower (forexample C₁₋₄) alkyl alcohols (for example methanol, ethanol oriso-propanol), ketones (such as acetone), esters (such as ethyl acetate)or mixtures thereof.

In one particular aspect of the invention R¹ is CHF₂ or CH₂CH₂F.

The variable n is preferably 0 or 2.

More preferred compounds of formula (I) include those in which nrepresents 0, or those in which n represents 2, so providing two fluoroatoms located at the 2- and 6-positions (that is the two ortho-positionsrelative to the point of attachment of the benzene ring to the —NH—CH₂—group).

The compound of formula (I) is especially Compound A, Compound B orCompound C.

Preferred salts of the compounds of formula (I) are acid addition salts.Acid addition salts include inorganic acid addition salts, such as thoseof sulphuric acid, nitric acid, phosphoric acid and hydrohalic acids,such as hydrobromic acid and hydrochloric acid. More preferred acidaddition salts include those of organic acids, such as those ofdimethylphosphoric acid; saccharinic acid; cyclohexylsulfamic acid;those of carboxylic acids (such as maleic acid, fumaric acid, asparticacid, succinic acid, malonic acid, acetic acid, benzoic acid,terephthalic acid, hippuric acid, 1-hydroxy-2-naphthoic acid, pamoicacid, hydroxybenzoic acid and the like); those of hydroxy acids (such assalicylic acid, tartaric acid, citric acid, malic acid (includingL-(−)-malic acid and, D,L-malic acid), gluconic acid (includingD-gluconic acid), glycolic acid, ascorbic acid, lactic acid and thelike); those of amino acids (such as glutamic acid (includingD-glutamic, L-glutamic, and D,L-glutamic, acids), arginine (includingL-arginine), lysine (including L-lysine and L-lysine hydrochloride),glycine and the like); and, particularly, those of sulfonic acids, (suchas 1,2-ethanedisulfonic acid, camphorsulfonic acids (including1S-(+)-10-camphorsulfonic acid and (+/−)-camphorsulfonic acids),ethanesulfonic acid, a propanesulfonic acid (including n-propanesulfonicacid), a butanesulfonic acid, a pentanesulfonic acid, a toluenesulfonicacid, methanesulfonic acid, p-xylenesulfonic acid, 2-mesitylenesulfonicacid, naphthalenesulfonic acids (including 1,5-naphthalenesulfonic acidand naphthalenesulfonic acid), benzenesulfonic acid,hydroxybenzenesulfonic acids, 2-hydroxyethanesulfonic acid,3-hydroxyethanesulfonic acid and the like).

Particularly preferred salts include those of C₁₋₆ (for example C₁₋₄)alkanesulfonic acids, such as ethanesulfonic acid (esylate) andpropanesulfonic acid (for example n-propanesulfonic acid) and optionallysubstituted (for example with one or more C₁₋₂ alkyl groups)arylsulfonic acids, such as benzenesulfonic acid (besylate) andnaphthalenedisulfonic acid.

Suitable stoichiometric ratios of acid to free base are in the range0.25:1.5 to 3.0:1, such as 0.45:1.25 to 1.25:1, including 0.50:1 to 1:1.

According to a further aspect of the invention there is providedformulation comprising a compound of formula (I) in substantiallycrystalline form.

Although we have found that it is possible to produce compounds of theinvention in forms which are greater than 80% crystalline, by“substantially crystalline” we include greater than 20%, preferablygreater than 30%, and more preferably greater than 40% (e.g. greaterthan any of 50, 60, 70, 80 or 90%) crystalline.

According to a further aspect of the invention there is also provided acompound of the invention in partially crystalline form. By “partiallycrystalline” we include 5% or between 5% and 20% crystalline.

The degree (%) of crystallinity may be determined by the skilled personusing X-ray powder diffraction (XRPD). Other techniques, such as solidstate NMR, FT-IR, Raman spectroscopy, differential scanning calorimetry(DSC) and microcalorimetry, may also be used.

Preferred compounds of formula (I) that may be prepared in crystallineform include salts of C₁₋₆ (for example C₂₋₆, such as C₂₋₄)alkanesulfonic acids, such as ethanesulfonic acid, propanesulfonic acid(for example n-propanesufonic acid) and optionally substitutedarylsulfonic acids, such as benzenesulfonic acid andnaphthalenedisulfonic acid.

The term “immediate release” pharmaceutical formulation includes anyformulation in which the rate of release of drug from the formulationand/or the absorption of drug, is neither appreciably, norintentionally, retarded by galenic manipulations. In the present case,immediate release may be provided for by way of an appropriatepharmaceutically acceptable diluent or carrier, which diluent or carrierdoes not prolong, to an appreciable extent, the rate of drug releaseand/or absorption. Thus, the term excludes formulations which areadapted to provide for “modified”, “controlled”, “sustained”,“prolonged”, “extended” or “delayed” release of drug.

In this context, the term “release” includes the provision (orpresentation) of drug from the formulation to the gastrointestinaltract, to body tissues and/or into systemic circulation. Forgastrointestinal tract release, the release is under pH conditions suchas pH=1 to 3, especially at, or about, pH=1. In one aspect of theinvention a formulation as described herein with a compound of formula(I), or an acid addition salt thereof, in crystalline form releases drugunder a range of pH conditions. In another aspect of the invention aformulation as described herein with a compound of formula (I), or anacid addition salt thereof, releases drug under pH conditions such aspH=1 to 3, especially at, or about, pH=1. Thus, formulations of theinvention may release at least 70% (preferably 80%) of active ingredientwithin 4 hours, such as within 3 hours, preferably 2 hours, morepreferably within 1.5 hours, and especially within an hour (such aswithin 30 minutes), of administration, whether this be oral orparenteral.

The formulations of the invention may be formulated in accordance with avariety of known techniques, for example as described by M. E. Aulton in“Pharmaceutics: The Science of Dosage Form Design” (1988) (ChurchillLivingstone), the relevant disclosures in which document are herebyincorporated by reference.

Formulations of the invention may be, or may be adapted in accordancewith standard techniques to be, suitable for peroral administration, forexample in the form of an immediate release tablet, an immediate releasecapsule or as a liquid dosage form, comprising active ingredient. Theseformulation types are well known to the skilled person and may beprepared in accordance with techniques known in the art.

Suitable diluents/carriers (which may also be termed “fillers”) for usein peroral formulations of the invention, for example those in the formof immediate release tablets, include monobasic calcium phosphate,dibasic calcium phosphate (including dibasic calcium phosphate dihydrateand dibasic calcium phosphate anhydrate), tribasic calcium phosphate,lactose, microcrystalline cellulose, silicified microcrystallinecellulose, mannitol, sorbitol, starch (such as maize, potato or rice),glucose, calcium lactate, calcium carbonate and the like. Preferreddiluents/carriers include dibasic calcium phosphate and microcrystallinecellulose, which may be used alone or in combination with anotherdiluent/carrier such as mannitol.

A formulation of the invention in the form of an immediate releasetablet may comprise one or more excipients to improve the physicaland/or chemical properties of the final composition, and/or tofacilitate the process of manufacture. Such excipients are conventionalin the formulation of immediate release formulations for peroral drugdelivery, and include one or more of the following: one or morelubricants (such as magnesium stearate, stearic acid, calcium stearate,stearyl alcohol or, preferably, sodium stearyl fumarate); a glidant(such as talc or a colloidal silica); one or more binders (such aspolyvinylpyrrolidone, microcrystalline cellulose, a polyethylene glycol(PEG), a polyethylene oxide, a hydroxypropylmethylcellulose (HPMC) of alow molecular weight, a methylcellulose (MC) of a low molecular weight,a hydroxypropylcellulose (HPC) of a low molecular weight, ahydroxyethylcellulose (HEC) of a low molecular weight, a starch (such asmaize, potato or rice) or a sodium carboxymethyl cellulose of a lowmolecular weight; (preferred binders are polyvinylpyrrolidone or a HPMCof a low molecular weight); one or more pH controlling agents (such asan organic acid (for example citric acid) or an alkali metal (forexample sodium) salt thereof, an oxide of magnesium, an alkali oralkaline earth metal (for example sodium, calcium or potassium)sulphate, metabisulphate, propionate or sorbate); one or moredisintegrant (for example sodium starch glycollate, a crosslinkedpolyvinylpyrrolidone, a crosslinked sodium carboxymethyl cellulose, astarch (such as maize, potato or rice) or an alginate); a colourant, aflavouring, a tonicity-modifying agent, a coating agent or apreservative.

It will be appreciated that some of the above mentioned excipients whichmay be present in a final immediate release oral (for example tablet)formulation of the invention may have more than one of the above-statedfunctions.

In a further aspect of the invention a liquid formulation of theinvention is adapted to be suitable for oral administration.

Suitable liquid formulations that are to be administered orally includethose in which a compound of formula (I) especially Compound A, CompoundB or Compound C, or a pharmaceutically acceptable salt thereof ispresented together with an aqueous carrier, such as water. It will benoted however, that certain specific formulations are not claimed (seeparticular aspects and the claims).

A formulation of the present invention comprising an aqueous carrier mayfurther comprise one or more excipients, such as an antimicrobialpreservative; a tonicity modifier (for example sodium chloride, mannitolor glucose); a pH adjusting agent (for example a common inorganic acidor base, including hydrochloric acid or sodium hydroxide); a pHcontrolling agents (that is, a buffer; for example tartaric acid, aceticacid or citric acid); a surfactant (for example Sodiun dodecyl sulphate(SDS) or Solutol™); a solubiliser which serves to help solubilise theactive ingredient (for example ethanol, a polyethylene glycol orhydroxypropyl-β-cyclodextrin or polyvinyl chloride (PVP)); or anantioxidant.

Liquid oral formulations may be in the form of suspensions of activeingredient in association with an aqueous solvent or, more preferablyaqueous solutions (that is, solutions of active compound including wateras a solvent). In this context, the term “aqueous solution” includesformulations in which at least 99% of active ingredient is in solutionat above 5° C. and atmospheric pressure, and the term “suspension” meansthat more than 1% of active ingredient is not in solution under suchconditions. Typical dispersion agents for suspensions are hydroxypropylmethylcellulose, AOT (dioctylsulfosuccinate), PVP and SDS. Otheralternatives may be possible.

In another aspect the present invention provides a liquid oralformulation comprising a compound of formula (I), or a pharmaceuticallyacceptable salt thereof, water and at least one additional agent. Theadditional agents include:

-   -   i. polyethylene glycol (PEG) and optionally also ethanol and/or        tartaric acid and/or citric acid and/or hydrochloric acid; or    -   ii. sodium chloride (which will be dissolved in the        formulation), and optionally also ethanol; or    -   iii. hydrochloric acid and/or sodium hydroxide to bring the pH        to a suitable value (preferably in the range 3-8 for a compound        of formula (I) wherein R² is methoxy or ethoxy, such as Compound        A, B or C); or    -   iv. DMA (dimethyl acetamide) and optionally also a medium chain        triglyceride (such as miglyol); or    -   v. a β-cyclodextrin (such as hydroxypropyl-β-cyclodextrin);    -   vi. a tonicity modifier such as sodium chloride and/or mannitol.

In a further aspect the present invention provides an oral solutioncomprising a compound of formula (I), or a pharmaceutically acceptablesalt thereof, (preferably Compound A, B or C) water and at least oneadditional agents as recited in (i) to (vi) above.

In another aspect the invention provides an aqueous formulation of acompound of formula (I) (such as Compound A, B or C) comprising asolubilising agent such as a polyethylene glycol, β-cyclodextrin (suchas hydroxypropyl-β-cyclodextrin), sorbitol or ethanol.

In a further aspect the present invention provides an oral solutionformulation comprising a compound of formula (I) and ethanol. Thisformulation can further comprise a medium chain triglyceride (such asmiglyol).

In a still further aspect the present invention provides an oralsolution formulation comprising a compound of formula (I) and DMA. Thisformulation can further comprise a medium chain triglyceride (such asmiglyol).

In another aspect the compound of formula (I) is crystalline (especiallya salt of Compound A; preferably a C₁₋₆ (for example C₂₋₆, such as C₂₋₄)alkanesulfonic acid salt, such as ethanesulfonic acid, propanesulfonicacid (for example n-propanesufonic acid) or an optionally substitutedarylsulfonic acid salt, such as benzenesulfonic acid ornaphthalenedisulfonic acid salt).

A particular liquid immediate release oral pharmaceutical formulation asclaimed in claim 1 is provided wherein the active ingredient is:

-   Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(OMe),-   Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(OMe),-   Ph(3-Cl)(5-OCH₂CH₂F)—(R)CH(OH)C(O)—(S)Aze-Pab(OMe),    or a pharmaceutically acceptable salt thereof.

A further particular liquid immediate release oral pharmaceuticalformulation as claimed in claim 1 is provided wherein the activeingredient is: Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(OMe) or a C₁₋₄alkanesulfonic acid or an optionally substituted arylsulfonic acid saltthereof

A yet further particular liquid immediate release oral pharmaceuticalformulation as claimed in claim 1 is provided wherein the activeingredient is: Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(OMe)or an optionally substituted arylsulfonic acid salt thereof (such as thenaphthalene-1,5-disulphonic acid salt).

It will be noted however, that certain specific formulations are notclaimed (see particular aspects and the claims).

In a further aspect of the invention a formulation of the invention isadapted to be suitable for parenteral administration. The term“parenteral” includes any mode of administration that does not compriseperoral administration to the gastrointestinal tract and includesadministration subcutaneously, intravenously, intraarterially,transdermally, intranasally, intrabuccally, intracutaneously,intramuscularly, intralipomateously, intraperitoneally, rectally,sublingually, topically, by inhalation, or by any other parenteralroute.

Suitable formulations of the invention that are to be administeredparenterally include those in which a compound of formula (I) or apharmaceutically acceptable salt thereof is presented together with anaqueous carrier, such as water.

A formulation of the present invention comprising an aqueous carrier mayfurther comprise one or more excipients, such as an antimicrobialpreservative; a tonicity modifier (for example sodium chloride, mannitolor glucose); a pH adjusting agent (for example a common inorganic acidor base, including hydrochloric acid or sodium hydroxide); a pHcontrolling agents (that is, a buffer; for example tartaric acid, aceticacid or citric acid); a surfactant (for example sodium dodecyl sulphate(SDS) or Solutol™); a solubiliser which serves to help solubilise theactive ingredient (for example ethanol, a polyethylene glycol orhydroxypropyl-β-cyclodextrin or polyvinyl chloride (PVP)); or anantioxidant.

Parenteral formulations may be in the form of suspensions of activeingredient in association with an aqueous solvent or, more preferablyaqueous solutions (that is, solutions of active compound including wateras a solvent). In this context, the term “aqueous solution” includesformulations in which at least 99% of active ingredient is in solutionat above 5° C. and atmospheric pressure, and the term “suspension” meansthat more than 1% of active ingredient is not in solution under suchconditions. Typical dispersion agents for suspensions are hydroxypropylmethylcellulose, AOT, PVP and SDS, but other alternatives are possible.

The number of excipients employed in the peroral and parenteralformulations of the invention depends upon many factors, such as thenature and amount of active ingredient present, and the amount ofdiluent/carrier (aqueous solvent or otherwise) that is included.

In another aspect the present invention provides a parenteralformulation comprising a compound of formula (I), or a pharmaceuticallyacceptable salt thereof, water and at least one additional agents. Theadditional agents include:

-   -   i. polyethylene glycol (PEG) and optionally also ethanol and/or        tartaric acid and/or hydrochloric acid; or    -   ii. sodium chloride (which will be dissolved in the        formulation), and optionally also ethanol; or    -   iii. hydrochloric acid and/or sodium hydroxide to bring the pH        to a suitable value (preferably in the range 3-8 for a compound        of formula (I) wherein R² is hydrogen, such as Compound D, E or        F; or preferably in the range 3.5-8 for a compound of        formula (I) wherein R² is methoxy or ethoxy, such as Compound A,        B or C); or    -   iv. DMA (dimethyl acetamide) and optionally also a medium chain        triglyceride (such as miglyol); or    -   v. a β-cyclodextrin (such as hydroxypropyl-β-cyclodextrin);    -   vi. a tonicity modifier such as sodium chloride and/or mannitol.

In a further aspect the present invention provides an injectablesolution comprising a compound of formula (I), or a pharmaceuticallyacceptable salt thereof, (preferably Compound D, E or F) water and atleast one additional agents as recited in (i) to (vi) above.

In another aspect the invention provides an aqueous formulation of acompound of formula (I) (such as Compound D, E or F) comprising asolubilising agent such as a polyethylene glycol, β-cyclodextrin (suchas hydroxypropyl-β-cyclodextrin), sorbitol or ethanol.

In a further aspect the present invention provides a parenteralformulation comprising a compound of formula (I) and ethanol. Thisformulation can further comprise a medium chain triglyceride (such asmiglyol).

In a still further aspect the present invention provides a parenteralformulation comprising a compound of formula (I) and DMA. Thisformulation can further comprise a medium chain triglyceride (such asmiglyol).

In another aspect the compound of formula (I) is crystalline (especiallya salt of Compound A; preferably a C₁₋₆ (for example C₂₋₆, such as C₂₋₄)alkanesulfonic acid salt, such as ethanesulfonic acid, propanesulfonicacid (for example n-propanesufonic acid) or an optionally substitutedarylsulfonic acid salt, such as benzenesulfonic acid salt).

In yet another aspect the formulation of the present invention is in asolid dosage form wherein R² is hydroxy, methoxy or ethoxy (preferablymethoxy) (the compound of formula (I) is especially Compound A, CompoundB or Compound C).

In yet another aspect the present invention provides a parenteralformulation (especially a water-based, injectable solution) comprising acompound of formula (I) in free base form.

In a further aspect the present invention provides a parenteralformulation comprising a compound of formula (I) in free base formwherein R² is hydrogen.

In a still further aspect the present invention provides a solidformulation comprising microcrystalline cellulose and polyvinylpyrrolidone (PVP); or comprising microcrystalline cellulose and sodiumstarch glycollate.

Formulations of the invention, such as parenteral formulations,comprising salts may be prepared by addition of diluent/carrier to theappropriate pre-prepared salt.

Compositions including active ingredient may also be provided in solidform suitable for use in the preparation of a formulation of theinvention (for example a solution, such as an aqueous solution, forexample for parenteral adminstration) ex tempore. Such compositions maybe in the form of a solid comprising active ingredient, optionally inthe presence of one or more further excipients as hereinbefore definedand, optionally, up to 10% (w/w) of diluent and/or carrier ashereinbefore defined, which compositions are hereinafter referred to as“the solid compositions of the invention”.

Solid compositions of the invention may be made by removal ofdiluent/carrier (for example solvent) from a formulation of theinvention, or a concentrated formulation of the invention, which may forexample be in the form of a solution, such as an aqueous solution.

In another aspect the present invention provides an orallyadministerable, immediate release formulation comprising a compound offormula (I), or a salt thereof, a carrier (such as microcrystallinecellulose), a disintegrant (such as sodium starch glycollate), a binder(such as polyvinyl pyrrolidone) and a lubricant (such as sodium stearylfumarate). Such a formulation may also comprise an additional carrier(or filler) such as mannitol.

Formulations of the invention that are in the form of immediate releasetablets may be prepared by bringing active ingredient into associationwith diluent/carrier using standard techniques, and using standardequipment, known to the skilled person, including wet or drygranulation, direct compression/compaction, drying, milling, mixing,tableting and coating, as well as combinations of these processes, forexample as described hereinafter. In one aspect of the invention, acidaddition salts of compounds of formula (I) in crystalline form areformulated in tablets.

There is thus provided a process for the formation of a solidcomposition suitable for use in the preparation of a formulation of theinvention (for example a solution, such as an aqueous solution) extempore, which process comprises removal of diluent/carrier (for examplesolvent) from a formulation of the invention, or a concentratedformulation of the invention.

Solvent may be removed by way of a variety of techniques known to thoseskilled in the art, for example evaporation (under reduced pressure orotherwise), freeze-drying, or any solvent removal (drying) process thatremoves solvent (such as water) while maintaining the integrity of theactive ingredient. An example of drying is freeze-drying.

Thus according to a further aspect of the invention there is provided afreeze-dried (lyophilised) solid composition of the invention.

In the preparation of solid compositions of the invention, the skilledperson will appreciate that appropriate additional excipients may beadded at a suitable stage prior to removal of diluent/carrier. Forexample, in the case of aqueous solutions, pH may be controlled and/oradjusted as hereinbefore described. Furthermore, an appropriateadditional excipient may be added with a view to aiding the formation ofa solid composition of the invention during the process ofdiluent/carrier removal (for example mannitol, sucrose, glucose, mannoseor trehalose).

A solid composition of a compound of formula (I) or a salt thereof, thusincludes a composition in which the solvent (for example water) content,other than a solvent of crystallization, is no more than 10%, such asless than 2% unbound solvent, such as water.

Formulations of the invention may be sterilised, for example by sterilefiltration or autoclavation, and/or filled into primary packages, suchas vials, cartridges and pre-filled syringes. Such processing steps mayalso take place prior to drying to form a solid composition of theinvention.

Before administration, the dried solid composition may be reconstitutedand/or diluted in, for instance, water, physiological saline, glucosesolution or any other suitable solution.

The amount of diluent/carrier in an oral (for example immediate releasetablet) formulation of the invention depends upon many factors, such asthe nature and amount of the active ingredient that is employed and thenature, and amounts, of any other constituents (for example furtherexcipients) that are present in the formulation, but is typically up to40% (w/w), preferably up to 30%, more preferably up to 20%, andparticularly up to 10% (w/w) of the final composition. The amount ofadditional excipients in such an oral formulation of the invention alsodepends upon factors, such as the nature and amount of the activeingredient that is employed, as well as the nature, and amounts, of anyother constituents (for example diluents/carriers and/or other furtherexcipients) that are present in the formulation, but, for lubricants andglidants is typically up to 5% (w/w), and for binders and disintegrantsis typically up to 10% (w/w) of the final composition.

The formulations of the invention are administered to mammalian patients(including humans), and, for compounds of formula (I) wherein R² is nothydrogen, are thereafter metabolised in the body to form compounds offormula (I) wherein R² is hydrogen that are pharmacologically active.

According to a further aspect of the invention there is thus provided aformulation of the invention for use as a pharmaceutical.

In particular, the compounds of formula (I) are, or are metabolisedfollowing administration to form, potent inhibitors of thrombin, forexample as may be demonstrated in the tests described in inter aliainternational patent application No. PCT/SE01/02657, as well asinternational patent applications WO 02/14270, WO 01/87879 and WO00/42059, the relevant disclosures in which documents are herebyincorporated by reference.

By “prodrug of a thrombin inhibitor”, we include compounds that aremetabolised following administration and form a thrombin inhibitor, inan experimentally-detectable amount, following administration.

By “active ingredient” and “active substance” we mean the pharmaceuticalagent (covering thrombin inhibitor and prodrugs thereof) present in theformulation.

The formulations of the invention are thus expected to be useful inthose conditions where inhibition of thrombin is required, and/orconditions where anticoagulant therapy is indicated, including thefollowing:

The treatment and/or prophylaxis of thrombosis and hypercoagulability inblood and/or tissues of animals including man. It is known thathypercoagulability may lead to thrombo-embolic diseases. Conditionsassociated with hypercoagulability and thrombo-embolic diseases whichmay be mentioned include inherited or acquired activated protein Cresistance, such as the factor V-mutation (factor V Leiden), andinherited or acquired deficiencies in antithrombin III, protein C,protein S, heparin cofactor II. Other conditions known to be associatedwith hypercoagulability and thrombo-embolic disease include circulatingantiphospholipid antibodies (Lupus anticoagulant), homocysteinemi,heparin induced thrombocytopenia and defects in fibrinolysis, as well ascoagulation syndromes (for example disseminated intravascularcoagulation (DIC)) and vascular injury in general (for example due tosurgery).

The treatment of conditions where there is an undesirable excess ofthrombin without signs of hypercoagulability, for example inneurodegenerative diseases such as Alzheimer's disease.

Particular disease states which may be mentioned include the therapeuticand/or prophylactic treatment of venous thrombosis (for example DVT) andpulmonary embolism, arterial thrombosis (e.g. in myocardial infarction,unstable angina, thrombosis-based stroke and peripheral arterialthrombosis), and systemic embolism usually from the atrium during atrialfibrillation (for example non-valvular atrial fibrillation) or from theleft ventricle after transmural myocardial infarction, or caused bycongestive heart failure; prophylaxis of re-occlusion (that isthrombosis) after thrombolysis, percutaneous trans-luminal angioplasty(PTA) and coronary bypass operations; the prevention of re-thrombosisafter microsurgery and vascular surgery in general.

Further indications include the therapeutic and/or prophylactictreatment of disseminated intravascular coagulation caused by bacteria,multiple trauma, intoxication or any other mechanism; anticoagulanttreatment when blood is in contact with foreign surfaces in the bodysuch as vascular grafts, vascular stents, vascular catheters, mechanicaland biological prosthetic valves or any other medical device; andanticoagulant treatment when blood is in contact with medical devicesoutside the body such as during cardiovascular surgery using aheart-lung machine or in haemodialysis; the therapeutic and/orprophylactic treatment of idiopathic and adult respiratory distresssyndrome, pulmonary fibrosis following treatment with radiation orchemotherapy, septic shock, septicemia, inflammatory responses, whichinclude, but are not limited to, edema, acute or chronic atherosclerosissuch as coronary arterial disease and the formation of atheroscleroticplaques, cerebral arterial disease, cerebral infarction, cerebralthrombosis, cerebral embolism, peripheral arterial disease, ischaemia,angina (including unstable angina), reperfusion damage, restenosis afterpercutaneous trans-luminal angioplasty (PTA) and coronary artery bypasssurgery.

The formulation of the present invention may also comprise anyantithrombotic agent(s) with a different mechanism of action to that ofthe compounds of formula (I), such as one or more of the following: theantiplatelet agents acetylsalicylic acid, ticlopidine and clopidogrel;thromboxane receptor and/or synthetase inhibitors; fibrinogen receptorantagonists; prostacyclin mimetics; phosphodiesterase inhibitors;ADP-receptor (P₂T) antagonists; and inhibitors of carboxypeptidase U(CPU).

Compounds of formula (I) that inhibit trypsin and/or thrombin may alsobe useful in the treatment of pancreatitis.

The formulations of the invention are thus indicated both in thetherapeutic and/or prophylactic treatment of these conditions.

According to a further aspect of the present invention, there isprovided a method of treatment of a condition where inhibition ofthrombin is required which method comprises administration of atherapeutically effective amount of a formulation of the invention to aperson suffering from, or susceptible to, such a condition.

In a still further aspect the present invention provides a formulationof the invention in the manufacture of a medicament for use in thetreatment of thrombosis.

According to a further aspect of the invention, there is provided amethod of treatment of thrombosis which method comprises administrationof a formulation of the invention to a person suffering from, orsusceptible to, such a condition.

For the avoidance of doubt, by “treatment” we include the therapeutictreatment, as well as the prophylaxis, of a condition.

Suitable amounts of active ingredient in formulations (oral orparenteral), concentrated formulations, and solid compositions, of theinvention depend upon many factors, such as the nature of thatingredient (free base/salt etc), the dose that is required in an oralformulation or in a final “ready to use” parenteral formulation that is,or is to be, prepared, and the nature, and amounts, of otherconstituents of the formulation. However, a typical daily dose of acompound of formula (I), or a pharmaceutically acceptable salt thereof,is in the range 0.001-100 mg/kg body weight at peroral administrationand 0.001-50 mg/kg body weight at parenteral administration, excludingthe weight of any acid counter-ion, irrespective of the number ofindividual doses that are administered during the course of that day. Inthe case of an immediate release parenteral formulation administrationmay be continuous (for example by way of infusion). A preferred dailyoral dose is 20-500 mg and a preferred parenteral dose is in the range0.1-50 mg.

General Procedures

TLC was performed on silica gel. Chiral HPLC analysis was performedusing a 46 mm×250 mm Chiralcel OD column with a 5 cm guard column. Thecolumn temperature was maintained at 35° C. A flow rate of 1.0 mL/minwas used. A Gilson 115 UV detector at 228 nm was used. The mobile phaseconsisted of hexanes, ethanol and trifluroacetic acid and theappropriate ratios are listed for each compound. Typically, the productwas dissolved in a minimal amount of ethanol and this was diluted withthe mobile phase.

In Preparations A to I below, LC-MS/MS was performed using a HP-1100instrument equipped with a CTC-PAL injector and a 5 Tm, 4×100 mmThermoQuest, Hypersil BDS-C18 column. An API-3000 (Sciex) MS detectorwas used. The flow rate was 1.2 mL/min and the mobile phase (gradient)consisted of 10-90% acetonitrile with 90-10% of 4 mM aq. ammoniumacetate, both containing 0.2% formic acid. Otherwise, low resolutionmass spectra (LRMS) were recorded using a Micromass ZQ spectrometer inESI posneg switching ion mode (mass range m/z 100-800); and highresolution mass spectra (HRMS) were recorded using a Micromass LCTspectrometer in ES negative ionization mode (mass range m/z 100-1000)with Leucine Enkephalin (C₂₈H₃₇N₅O₇) as internal mass standard.

¹H NMR spectra were recorded using tetramethylsilane as the internalstandard.

Processes for the synthesis of compounds of formula (I) are contained inInternational Patent Application No. PCT/SE01/02657 (WO 02/44145,earliest priority date 1 Dec. 2000, filed 30 Nov. 2001, published 6 Jun.2002)), relevant information from which is incorporated herein.

Preparation A: Preparation of Compound A

(i) 3-Chloro-5-methoxybenzaldehyde

3,5-Dichloroanisole (74.0 g, 419 mmol) in THF (200 mL) was addeddropwise to magnesium metal (14.2 g, 585 mmol, pre-washed with 0.5 NHCl) in THF (100 mL) at 25° C. After the addition, 1,2-dibromoethane(3.9 g, 20.8 mmol) was added dropwise. The resultant dark brown mixturewas heated at reflux for 3 h. The mixture was cooled to 0° C., andN,N-dimethylformamide (60 mL) was added in one portion. The mixture waspartitioned with diethyl ether (3×400 mL) and 6N HCl (500 mL). Thecombined organic extracts were washed with brine (300 mL), dried(Na₂SO₄), filtered and concentrated in vacuo to give an oil. Flashchromatography (2×) on silica gel eluting with Hex:EtOAc (4:1) affordedthe sub-title compound (38.9 g, 54%) as a yellow oil.

¹H NMR (300 MHz, CDCl₃) δ 9.90 (s, 1H), 7.53 (s, 1H), 7.38 (s, 1H), 7.15(s, 1H), 3.87 (s, 3H).

(ii) 3-Chloro-5-hydroxybenzaldehyde

A solution of 3-chloro-5-methoxybenzaldehyde (22.8 g, 134 mmol; see step(i) above) in CH₂Cl₂ (250 mL) was cooled to 0° C. Boron tribromide (15.8mL, 167 mmol) was added dropwise over 15 min. After stirring, thereaction mixture for 2 h, H₂O (50 mL) was added slowly. The solution wasthen extracted with Et₂O (2×100 mL). The organic layers were combined,dried (Na₂SO₄), filtered and concentrated in vacuo. Flash chromatographyon silica gel eluting with Hex:EtOAc (4:1) afforded the sub-titlecompound (5.2 g, 25%).

¹H NMR (300 MHz, CDCl₃) δ 9.85 (s, 1H), 7.35 (s, 1H), 7.20 (s, 1H), 7.10(s, 1H), 3.68 (s, 1H)

(iii) 3-Chloro-5-difluoromethoxybenzaldehyde

A solution of 3-chloro-5-hydroxybenzaldehyde (7.5 g, 48 mmol; see step(ii) above) in 2-propanol (250 mL) and 30% KOH (100 mL) was heated toreflux. While stirring, CHClF₂ was bubbled into the reaction mixture for2 h. The reaction mixture was cooled, acidified with 1N HCl andextracted with EtOAc (2×100 mL). The organics were washed with brine(100 mL), dried (Na₂SO₄), filtered and concentrated in vacuo. Flashchromatography on silica gel eluting with Hex:EtOAc (4:1) afforded thesub-title compound (4.6 g, 46%).

¹H NMR (300 MHz, CDCl₃) δ 9.95 (s, 1H), 7.72 (s, 1H), 7.52 (s, 1H), 7.40(s, 1H), 6.60 (t, J_(H-F)=71.1 Hz, 1H)

(iv) Ph(3-Cl)(5-OCHF₂)—(R,S)CH(OTMS)CN

A solution of 3-chloro-5-difluoromethoxybenzaldehyde (4.6 g, 22.3 mmol;see step (iii) above) in CH₂Cl₂ (200 mL) was cooled to 0° C. ZnI₂ (1.8g, 5.6 mmol) and trimethylsilyl cyanide (2.8 g, 27.9 mmol) were addedand the reaction mixture was allowed to warm to room temperature andstirred for 15 h. The mixture was partially concentrated in vacuoyielding the sub-title compound as a liquid, which was used directly instep (v) below without further purification or characterization.

(v) Ph(3-Cl)(5-OCHF₂)—(R,S)CH(OH)C(NH)OEt

Ph(3-Cl)(5-OCHF₂)—(R,S)CH(OTMS)CN (6.82 g, assume 22.3 mmol; see step(iv) above) was added dropwise to HCl/EtOH (500 mL). The reactionmixture was stirred 15 h, then partially concentrated in vacuo yieldingthe sub-title compound as a liquid, which was used in step (vi) withoutfurther purification or characterization.

(vi) Ph(3-Cl)(5-OCHF₂)—(R,S)CH(OH)C(O)OEt

Ph(3-Cl)(5-OCHF₂)—(R,S)CH(OH)C(NH)OEt (6.24 g, assume 22.3 mmol; seestep (v) above) was dissolved in THF (250 mL), 0.5M H₂SO₄ (400 mL) wasadded and the reaction was stirred at 40° C. for 65 h, cooled and thenpartially concentrated in vacuo to remove most of the THF. The reactionmixture was then extracted with Et₂O (3×100 mL), dried (Na₂SO₄),filtered and concentrated in vacuo to afford the sub-title compound as asolid, which was used in step (vii) without further purification orcharacterization.

(vii) Ph(3-Cl)(5-OCHF₂)—(R,S)CH(OH)C(O)OH

A solution of Ph(3-Cl)(5-OCHF₂)—(R,S)CH(OH)C(O)OEt (6.25 g, assume 22.3mmol; see step (vi) above) in 2-propanol (175 mL) and 20% KOH (350 mL)was stirred at room temperature 15 h. The reaction was then partiallyconcentrated in vacuo to remove most of the 2-propanol. The remainingmixture was acidified with 1M H₂SO₄, extracted with Et₂O (3×100 mL),dried (Na₂SO₄) and concentrated in vacuo to give a solid. Flashchromatography on silica gel eluting with CHCl₃:MeOH:concentrated NH₄OH(6:3:1) afforded the ammonium salt of the sub-title compound. Theammonium salt was then dissolved in a mixture of EtOAc (75 mL) and H₂O(75 mL) and acidified with 2N HCl. The organic layer was separated andwashed with brine (50 mL), dried (Na₂SO₄) and concentrated in vacuo toafford the sub-title compound (3.2 g, 57% from steps (iv) to (vii)).

¹H NMR (300 MHz, CD₃OD) δ 7.38 (s, 1H), 7.22 (s, 1H), 7.15 (s, 1H), 6.89(t, J_(H-F)=71.1 Hz, 1H), 5.16 (s, 1H)

(viii) Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)OH (a) andPh(3-Cl)(5-OCHF₂)—(S)CH(OAc)C(O)OH (b)

A mixture of Ph(3-Cl)(5-OCHF₂)—(R,S)CH(OH)C(O)OH (3.2 g, 12.7 mmol; seestep (vii) above) and Lipase PS “Amano” (˜2.0 g) in vinyl acetate (125mL) and MTBE (125 mL) was heated at reflux for 48 h. The reactionmixture was cooled, filtered through Celite® and the filter cake washedwith EtOAc. The filtrate was concentrated in vacuo and subjected toflash chromatography on silica gel eluting with CHCl₃:MeOH:concentratedNH₄OH (6:3:1) yielding the ammonium salts of the sub-title compounds (a)and (b). Compound (a) as a salt was dissolved in H₂O, acidified with 2NHCl and extracted with EtOAc. The organic layer was washed with brine,dried (Na₂SO₄), filtered and concentrated in vacuo to afford thesub-title compound (a) (1.2 g, 37%).

For Sub-Title Compound (a)

¹H NMR (300 MHz, CD₃OD) δ 7.38 (s, 1H), 7.22 (s, 1H), 7.15 (s, 1H), 6.89(t, J_(H-F)=71.1 Hz, 1H), 5.17 (s, 1H)

(ix) Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab(Teoc)

To a solution of Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)OH (1.1 g, 4.4 mmol; seestep (viii) above) and H-Aze-Pab(Teoc) (see international patentapplication WO 00/42059, 2.6 g, 5.7 mmol) in DMF (50 mL) at 0° C. wasadded PyBOP (2.8 g, 5.3 mmol) and collidine (1.3 g, 10.6 mmol). Thereaction was stirred at 0° C. for 2 h and then at room temperature foran additional 15 h. The reaction mixture was concentrated in vacuo andflash chromatographed on silica gel (3×), eluting first with CHCl₃:EtOH(9:1), then with EtOAc:EtOH (20:1) and finally eluting with CH₂Cl₂:CH₃OH(95:5) to afford the sub-title compound (1.0 g, 37%) as a white solid.

¹H NMR (300 MHz, CD₃OD, mixture of rotamers) δ 7.79-7.85 (d, J=8.7 Hz,2H), 7.15-7.48 (m, 5H), 6.89 and 6.91 (t, J_(H-F)=71.1 Hz, 1H), 5.12 and5.20 (s, 1H), 4.75-4.85 (m, 1H), 3.97-4.55 (m, 6H), 2.10-2.75 (m, 2H),1.05-1.15 (m, 2H), 0.09 (s, 9H)

MS (m/z) 611 (M+1)⁺

(x) Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab(OMe, Teoc)

Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab(Teoc) (0.40 g, 0.65 mmol; seestep (ix) above), was dissolved in 20 mL of acetonitrile and 0.50 g (6.0mmol) of O-methyl hydroxylamine hydrochloride was added. The mixture washeated at 70° C. for 2 h. The solvent was evaporated and the residue waspartitioned between water and ethyl acetate. The aqueous phase wasextracted twice more with ethyl acetate and the combined organic phasewas washed with water, brine, dried (Na₂SO₄), filtered and evaporated.Yield: 0.41 g (91%).

¹H-NMR (400 MHz; CDCl₃): δ 7.83 (bt, 1H), 7.57 (bs, 1H), 7.47 (d, 2H),7.30 (d, 2H), 7.20 (m, 1H), 7.14 (m, 1H), 7.01 (m, 1H), 6.53 (t, 1H),4.89 (s, 1H), 4.87 (m, 1H), 4.47 (m, 2H), 4.4-4.2 (b, 1H), 4.17-4.1 (m,3H), 3.95 (s, 3H), 3.67 (m, 1H), 2.68 (m, 1H), 2.42 (m, 1H) 0.97 (m,2H), 0.01 (s, 9H).

(xi) Compound A

Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab(OMe, Teoc) (0.40 g, 0.62 mmol;see step (x) above), was dissolved in 5 mL of TFA and allowed to reactfor 30 min. TFA was evaporated and the residue was partitioned betweenethyl acetate and NaHCO₃ (aq.). The aqueous phase was extracted twicemore with ethyl acetate and the combined organic phase was washed withwater, brine, dried (Na₂SO₄), filtered and evaporated. The product wasfreeze dried from water/acetonitrile. No purification was necessary.Yield: 0.28 g (85%).

¹H-NMR (600 MHz; CDCl₃): δ 7.89 (bt, 1H), 7.57 (d, 2H), 7.28 (d, 2H),7.18 (m, 1H), 7.13 (m, 1H), 6.99 (m, 1H), 6.51 (t, 1H), 4.88 (s, 1H),4.87 (m, 1H), 4.80 (bs, 2H), 4.48 (dd, 1H), 4.43 (dd, 1H), 4.10 (m, 1H),3.89 (s, 3H), 3.68 (m, 1H), 2.68 (m, 1H), 2.40 (m, 1H).

¹³C-NMR (125 MHz; CDCl₃): (carbonyl and/or amidine carbons, rotamers) δ172.9, 170.8, 152.7, 152.6

HRMS calculated for C₂₂H₂₃ClF₂N₄O₅ (M−H)⁻ 495.1242, found 495.1247

Preparation B: Preparation of Compound B

(i) 2,6-Difluoro-4[(methylsulfinyl)(methylthio)methyl]benzonitrile

(Methylsulfinyl)(methylthio)methane (7.26 g, 0.0584 mol) was dissolvedin 100 mL of dry THF under argon and was cooled to −78° C. Butyllithiumin hexane (16 mL 1.6M, 0.0256 mol) was added dropwise with stirring. Themixture was stirred for 15 min. Meanwhile, a solution of3,4,5-trifluorobenzonitrile (4.0 g, 0.025 mmol) in 100 mL of dry THF wascooled to −78° C. under argon and the former solution was added througha cannula to the latter solution over a period of 35 min. After 30 min,the cooling bath was removed and when the reaction had reached roomtemperature it was poured into 400 mL of water. The THF was evaporatedand the remaining aqueous layer was extracted three times with diethylether. The combined ether phase was washed with water, dried (Na₂SO₄)and evaporated. Yield: 2.0 g(30%).

¹H NMR (500 MHz, CDCl₃) δ 7.4-7.25 (m, 2H), 5.01 (s, 1H, diasteromer),4.91 (s, 1H, diasteromer), 2.88 (s, 3H, diasteromer), 2.52 (s, 3H,diasteromer), 2.49 (s, 3H, diasteromer), 2.34 (s, 3H, diasteromer), 1.72(broad, 1H)

(ii) 2,6-Difluoro-4-formylbenzonitrile

2,6-Difluoro-4[(methylsulfinyl)(methylthio)methyl]benzonitrile (2.17 g,8.32 mmol; see step (i) above) was dissolved in 90 mL of THF and 3.5 mLof concentrated sulfuric acid was added. The mixture was left at roomtemperature for 3 days and subsequently poured into 450 mL of water.Extraction three times with EtOAc followed and the combined etherealphase was washed twice with aqueous sodium bicarbonate and with brine,dried (Na₂SO₄) and evaporated. Yield: 1.36 g (98%). The position of theformyl group was established by ¹³C NMR. The signal from the fluorinatedcarbons at 162.7 ppm exhibited the expected coupling pattern with twocoupling constants in the order of 260 Hz and 6.3 Hz respectivelycorresponding to an ipso and a meta coupling from the fluorine atoms.

¹H NMR (400 MHz, CDCl₃) δ 10.35 (s, 1H), 7.33 (m, 2H)

(iii) 2,6-Difluoro-4-hydroxymethylbenzonitrile

2,6-Difluoro-4-formylbenzonitrile (1.36 g, 8.13 mmol; see step (ii)above) was dissolved in 25 mL of methanol and cooled on an ice bath.Sodium borohydride (0.307 g, 8.12 mmol) was added in portions withstirring and the reaction was left for 65 min. The solvent wasevaporated and the residue was partitioned between diethyl ether andaqueous sodium bicarbonate. The ethereal layer was washed with moreaqueous sodium bicarbonate and brine, dried (Na₂SO₄) and evaporated. Thecrude product crystallised soon and could be used without furtherpurification. Yield: 1.24 g (90%).

¹H NMR (400 MHz, CDCl₃) δ 7.24 (m, 2H), 4.81 (s, 2H), 2.10 (broad, 1H)

(iv) 4-Cyano-2,6-difluorobenzyl methanesulfonate

To an ice cooled solution of 2,6-difluoro-4-hydroxymethylbenzonitrile(1.24 g, 7.32 mmol; see step (iii) above) and methanesulfonyl chloride(0.93 g, 8.1 mmol) in 60 mL of methylene chloride was addedtriethylamine (0.81 g, 8.1 mmol) with stirring. After 3 h at 0° C., themixture was washed twice with 1M HCl and once with water, dried (Na₂SO₄)and evaporated. The product could be used without further purification.Yield: 1.61 g (89%).

¹H NMR (300 MHz, CDCl₃) δ 7.29 (m, 2H), 5.33 (s, 2H), 3.07 (s, 3H)

(v) 4-Azidomethyl-2,6-difluorobenzonitrile

A mixture of 4-cyano-2,6-difluorobenzyl methanesulfonate (1.61 g, 6.51mmol; see step (iv) above) and sodium azide (0.72 g, 0.0111 mol) in 10mL of water and 20 mL of DMF was stirred at room temperature overnight.The resultant was subsequently poured into 200 mL of water and extractedthree times with diethyl ether. The combined ethereal phase was washedfive times with water, dried (Na₂SO₄) and evaporated. A small sample wasevaporated for NMR purposes and the product crystallised. The rest wasevaporated cautiously but not until complete dryness. Yield(theoretically 1.26 g) was assumed to be almost quantitative based onNMR and analytical HPLC.

¹H NMR (400 MHz, CDCl₃) δ 7.29 (m, 2H), 4.46 (s, 2H)

(vi) 4-Aminomethyl-2,6-difluorobenzonitrile

This reaction was carried out according to the procedure described in J.Chem. Res. (M) (1992) 3128. To a suspension of 520 mg of 10% Pd/C (50%moisture) in 20 mL of water was added a solution of sodium borohydride(0.834 g, 0.0221 mol) in 20 mL of water. Some gas evolution resulted.4-Azidomethyl-2,6-difluorobenzonitrile (1.26 g, 6.49 mmol; see step (v)above) was dissolved in 50 mL of THF and added to the aqueous mixture onan ice bath over 15 min. The mixture was stirred for 4 h, whereafter 20mL of 2M HCl was added and the mixture was filtered through Celite. TheCelite was rinsed with more water and the combined aqueous phase waswashed with EtOAc and subsequently made alkaline with 2M NaOH.Extraction three times with methylene chloride followed and the combinedorganic phase was washed with water, dried (Na₂SO₄) and evaporated.Yield: 0.87 g (80%).

¹H NMR (400 MHz, CDCl₃) δ 7.20 (m, 2H), 3.96 (s, 2H), 1.51 (broad, 2H)

(vii) 2,6-Difluoro-4-tert-butoxycarbonylaminomethylbenzonitrile

A solution of 4-aminomethyl-2,6-difluorobenzonitrile (0.876 g, 5.21mmol; see step (vi) above) was dissolved in 50 mL of THF anddi-tert-butyl dicarbonate (1.14 g, 5.22 mmol) in 10 mL of THF was added.The mixture was stirred for 3.5 h. The THF was evaporated and theresidue was partitioned between water and EtOAc. The organic layer waswashed three times with 0.5 M HCl and water, dried (Na₂SO₄) andevaporated. The product could be used without further purification.Yield: 1.38 g (99%).

¹H NMR (300 MHz, CDCl₃) δ 7.21 (m, 2H), 4.95 (broad, 1H), 4.43 (broad,2H), 1.52 (s, 9H)

(viii) Boc-Pab(2,6-diF)(OH)

A mixture of 2,6-difluoro-4-tert-butoxycarbonylaminomethylbenzonitrile(1.38 g, 5.16 mmol; see step (vii) above), hydroxylamine hydrochloride(1.08 g, 0.0155 mol) and triethylamine (1.57 g, 0.0155 mol) in 20 mL ofethanol was stirred at room temperature for 36 h. The solvent wasevaporated and the residue was partitioned between water and methylenechloride. The organic layer was washed with water, dried (Na₂SO₄) andevaporated. The product could be used without further purification.Yield: 1.43 g (92%).

¹H NMR (500 MHz, CD₃OD) δ 7.14 (m, 2H), 4.97 (broad, 1H), 4.84 (broad,2H), 4.40 (broad, 2H), 1.43 (s, 9H)

(ix) Boc-Pab(2,6-diF)xHOAc

This reaction was carried out according to the procedure described byJudkins et al, Synth. Comm. (1998) 4351. Boc-Pab(2,6-diF)(OH) (1.32 g,4.37 mmol; see step (viii) above), acetic anhydride (0.477 g, 4.68 mmol)and 442 mg of 10% Pd/C (50% moisture) in 100 mL of acetic acid washydrogenated at 5 atm pressure for 3.5 h. The mixture was filteredthrough Celite, rinsed with ethanol and evaporated. The residue wasfreeze-dried from acetonitrile and water and a few drops of ethanol. Thesub-title product could be used without further purification. Yield:1.49 g (99%).

¹H NMR (400 MHz, CD₃OD) δ 7.45 (m, 2H), 4.34 (s, 2H), 1.90 (s, 3H), 1.40(s, 9H)

(x) Boc-Pab(2,6-diF)(Teoc)

To a solution of Boc-Pab(2,6-diF)xHOAc (1.56 g, 5.49 mmol; see step (ix)above) in 100 mL of THF and 1 mL of water was added2-(trimethylsilyl)ethyl p-nitrophenyl carbonate (1.67 g, 5.89 mmol). Asolution of potassium carbonate (1.57 g, 0.0114 mol) in 20 mL of waterwas added dropwise over 5 min. The mixture was stirred overnight. TheTHF was evaporated and the residue was partitioned between water andmethylene chloride. The aqueous layer was extracted with methylenechloride and the combined organic phase was washed twice with aqueoussodium bicarbonate, dried (Na₂SO₄) and evaporated. Flash chromatographyon silica gel with heptane/EtOAc=2/1 gave 1.71 g (73%) of pure compound.

¹H NMR (400 MHz, CDCl₃) δ 7.43 (m, 2H), 4.97 (broad, 1H), 4.41 (broad,2H), 4.24 (m, 2H), 1.41 (s, 9H), 1.11 (m, 2H), 0.06 (s, 9H)

(xi) Boc-Aze-Pab(2,6-diF)(Teoc)

Boc-Pab(2,6-diF)(Teoc) (1.009 g, 2.35 mmol; see step (x) above) wasdissolved in 50 mL of EtOAc saturated with HCl(g). The mixture was leftfor 10 min., evaporated and dissolved in 18 mL of DMF, and then cooledon an ice bath. Boc-Aze-OH (0.450 g, 2.24 mmol), PyBOP (1.24 g, 2.35mmol) and lastly diisopropylethyl amine (1.158 g, 8.96 mmol) were added.The reaction mixture was stirred for 2 h and then poured into 350 mL ofwater and extracted three times with EtOAc. The combined organic phasewas washed with brine, dried (Na₂SO₄) and evaporated. Flashchromatography on silica gel with heptane:EtOAc (1:3) gave 1.097 g (96%)of the desired compound.

¹H NMR (500 MHz, CDCl₃) δ 7.46 (m, 2H), 4.65-4.5 (m, 3H), 4.23 (m, 2H),3.87 (m, 1H), 3.74 (m, 1H), 2.45-2.3 (m, 2H), 1.40 (s, 9H), 1.10 (m,2H), 0.05 (s, 9H)

(xii) Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab(2,6-diF)(Teoc)

Boc-Aze-Pab(2,6-diF)(Teoc) (0.256 g, 0.500 mmol; see step (xi) above)was dissolved in 20 mL of EtOAc saturated with HCl(g). The mixture wasleft for 10 min. and evaporated and dissolved in 5 mL of DMF.Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)OH (0.120 g, 0.475 mmol; see PreparationA(viii) above), PyBOP (0.263 g, 0.498 mmol) and lastly diisopropylethylamine (0.245 g, 1.89 mmol) were added. The reaction mixture was stirredfor 2 h and then poured into 350 mL of water and extracted three timeswith EtOAc. The combined organic phase was washed with brine, dried(Na₂SO₄) and evaporated. Flash chromatography on silica gel with EtOAcgave 0.184 g (60%) of the desired sub-title compound.

¹H NMR (400 MHz, CD₃OD, mixture of rotamers) δ 7.55-7.45 (m, 2H), 7.32(m, 1H, major rotamer), 7.27 (m, 1H, minor rotamer), 7.2-7.1 (m, 2H),6.90 (t, 1H, major rotamer), 6.86 (t, 1H, minor rotamer), 5.15 (s, 1H,major rotamer), 5.12 (m, 1H, minor rotamer), 5.06 (s, 1H, minorrotamer), 4.72 (m, 1H, major rotamer), 4.6-4.45 (m, 2H), 4.30 (m, 1H,major rotamer), 4.24 (m, 2H), 4.13 (m, 1H, major rotamer), 4.04 (m, 1H,minor rotamer), 3.95 (m, 1H, minor rotamer), 2.62 (m, 1H, minorrotamer), 2.48 (m, 1H, major rotamer), 2.22 (m, 1H, major rotamer), 2.10(m, 1H, minor rotamer), 1.07 (m, 2H), 0.07 (m, 9H)

(xiii) Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab(2,6-diF)(OMe,Teoc)

A mixture of Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab(2,6-diF)(Teoc) (64mg, 0.099 mmol; see step (xii) above) and O-methyl hydroxylaminehydrochloride (50 mg, 0.60 mmol) in 4 mL of acetonitrile was heated at70° C. for 3 h. The solvent was evaporated and the residue waspartitioned between water and EtOAc. The aqueous layer was extractedtwice with EtOAc and the combined organic phase was washed with water,dried (Na₂SO₄) and evaporated. The product could be used without furtherpurification. Yield: 58 mg (87%).

¹H NMR (400 MHz, CDCl₃) δ 7.90 (bt, 1H), 7.46 (m, 1H), 7.25-6.95 (m,5H), 6.51, t, 1H), 4.88 (s, 1H), 4.83 (m, 1H), 4.6-4.5 (m, 2H), 4.4-3.9(m, 4H), 3.95 (s, 3H), 3.63 (m, 1H), 2.67 (m, 1H), 2.38 (m, 1H), 1.87(broad, 1H), 0.98 (m, 2H), 0.01, s, 9H)

(xiv) Compound B

Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab(2,6-diF)(OMe,Teoc) (58 mg, 0.086mmol; see step (xiii) above) was dissolved in 3 mL of TFA, cooled on anice bath and allowed to react for 2 h. The TFA was evaporated and theresidue dissolved in EtOAc. The organic layer was washed twice withaqueous sodium carbonate and water, dried (Na₂SO₄) and evaporated. Theresidue was freeze-dried from water and acetonitrile to give 42 mg (92%)of the title compound.

¹H NMR (300 MHz, CDCl₃) δ 7.95 (bt, 1H), 7.2-7.1 (m, 4H), 6.99 (m, 1H),6.52 (t, 1H), 4.88 (s, 1H), 4.85-4.75 (m, 3H), 4.6-4.45 (m, 2H), 4.29(broad, 1H), 4.09 (m, 1H), 3.89 (s, 3H), 3.69 (m, 1H), 2.64 (m, 1H),2.38 (m, 1H), 1.85 (broad, 1H)

¹³C-NMR (100 MHz; CDCl₃): (carbonyl and/or amidine carbons) δ 172.1,169.8, 151.9

APCI-MS: (M+1)=533/535 m/z

Preparation C: Preparation of Compound C

(i) (2-Monofluoroethyl) methanesulfonate

To a magnetically stirred solution of 2-fluoroethanol (5.0 g, 78.0 mmol)in CH₂Cl₂ (90 mL) under nitrogen at 0° C. was added triethylamine (23.7g, 234 mmol) and methanesulfonyl chloride (10.7 g, 93.7 mmol). Themixture was stirred at 0° C. for 1.5 h, diluted with CH₂Cl₂ (100 mL) andwashed with 2N HCl (100 mL). The aqueous layer was extracted with CH₂Cl₂(50 mL) and the combined organic extracts washed with brine (75 mL),dried (Na₂SO₄), filtered and concentrated in vacuo to afford thesub-title compound (9.7 g, 88%) as a yellow oil which was used withoutfurther purification.

¹H NMR (300 MHz, CDCl₃) δ 4.76 (t, J=4 Hz, 1H), 4.64 (t, J=4 Hz, 1H),4.52 (t, J=4 Hz, 1H), 4.43 (t, J=4 Hz, 1H), 3.09 (s, 3H).

(ii) 3-Chloro-5-monofluoroethoxybenzaldehyde

To a solution of 3-chloro-5-hydroxybenzaldehyde (8.2 g, 52.5 mmol; seePreparation A(ii) above) and potassium carbonate (9.4 g, 68.2 mmol) inDMF (10 mL) under nitrogen was added a solution of (2-monofluoroethyl)methanesulfonate (9.7 g, 68.2 mmol; see step (i) above) in DMF (120 mL)dropwise at room temperature. The mixture was heated to 1001° C. for 5 hand then stirred overnight at room temperature. The reaction was cooledto 0° C., poured into ice-cold 2N HCl and extracted with EtOAc. Thecombined organic extracts were washed with brine, dried (Na₂SO₄),filtered and concentrated in vacuo. The brown oil was chromatographed onsilica gel eluting with Hex:EtOAc (4:1) to afford the sub-title compound(7.6 g, 71%) as a yellow oil.

¹H NMR (300 MHz, CDCl₃) δ 9.92 (s, 1H), 7.48 (s, 1H), 7.32 (s, 1H), 7.21(s, 1H), 4.87 (t, J=4 Hz, 1H), 4.71 (t, J=3 Hz, 1H), 4.33 (t, J=3 Hz,1H), 4.24 (t, J=3 Hz, 1H).

(iii) Ph(3-Cl)(5-OCH₂CH₂F)—(R,S)CH(OTMS)CN

To a solution of 3-chloro-5-monofluoroethoxybenzaldehyde (7.6 g, 37.5mmol; see step (ii) above) and zinc iodide (3.0 g, 9.38 mmol) in CH₂Cl₂(310 mL) was added trimethylsilyl cyanide (7.4 g, 75.0 mmol) dropwise at0° C. under nitrogen. The mixture was stirred at 0° C. for 3 h and atroom temperature overnight. The reaction was diluted with H₂O (300 mL),the organic layer was separated, dried (Na₂SO₄), filtered andconcentrated in vacuo to afford the sub-title compound (10.6 g, 94%) asa brown oil that was used without further purification orcharacterisation.

(iv) Ph(3-Cl)(5-OCH₂CH₂F)—(R,S)CH(OH)C(O)OH

Concentrated hydrochloric acid (100 mL) was added toPh(3-Cl)(5-OCH₂CH₂F)—(R,S)CH(OTMS)CN (10.6 g, 5.8 mmol; see step (iii)above) and the solution stirred at 100° C. for 3 h. After cooling toroom temperature, the reaction was further cooled to 0° C., basifiedslowly with 3N NaOH (300 mL) and washed with Et₂O (3×200 mL). Theaqueous layer was acidified with 2N HCl (80 mL) and extracted with EtOAc(3×300 mL). The combined EtOAc extracts were dried (Na₂SO₄), filteredand concentrated in vacuo to afford the sub-title compound (8.6 g, 98%)as a pale yellow solid that was used without further purification.

R_(f)=0.28 (90:8:2 CHCl₃:MeOH:concentrated NH₄OH)

¹H NMR (300 MHz, CD₃OD) δ 7.09 (s, 1H), 7.02 (s, 1H), 6.93 (s, 1H), 5.11(s, 1H), 4.77-4.81 (m, 1H), 4.62-4.65 (m, 1H), 4.25-4.28 (m, 1H),4.15-4.18 (m, 1H).

(v) Ph(3-Cl)(5-OCH₂CH₂F)—(S)CH(OAc)C(O)OH (a) andPh(3-Cl)(5-OCH₂CH₂F)—(R)CH(OH)C(O)OH (b)

A solution of Ph(3-Cl)(5-OCH₂CH₂F)—(R,S)CH(OH)C(O)OH (8.6 g, 34.5 mmol;see step (iv) above) and Lipase PS “Amano” (4.0 g) in vinyl acetate (250mL) and MTBE (250 mL) was heated at 70° C. under nitrogen for 3 d. Thereaction was cooled to room temperature and the enzyme removed byfiltration through Celite®. The filter cake was washed with EtOAc andthe filtrate concentrated in vacuo. Chromatography on silica gel elutingwith CHCl₃:MeOH:Et₃N (90:8:2) afforded the triethylamine salt ofsub-title compound (a) as a yellow oil. In addition, the triethylaminesalt of sub-title compound (b) (4.0 g) was obtained. The salt ofsub-title compound (b) was dissolved in H₂O (250 mL), acidified with 2NHCl and extracted with EtOAc (3×200 mL). The combined organic extractswere dried (Na₂SO₄), filtered and concentrated in vacuo to yield thesub-title compound (b) (2.8 g, 32%) as a yellow oil.

Data for Sub-Title Compound (b):

-   -   R_(f)=0.28 (90:8:2 CHCl₃:MeOH:concentrated NH₄OH)

¹H NMR (300 MHz, CD₃OD) δ 7.09 (s, 1H), 7.02 (s, 1H), 6.93 (s, 1H), 5.11(s, 1H), 4.77-4.81 (m, 1H), 4.62-4.65 (m, 1H), 4.25-4.28 (m, 1H),4.15-4.18 (m, 1H).

(vi) Compound C

To a solution of Ph(3-Cl)(5-OCH₂CH₂F)—(R)CH(OH)C(O)OH (818 mg, 3.29mmol; see step (v) above) in DMF (30 mL) under nitrogen at 0° C. wasadded HAze-Pab(OMe).2HCl (1.43 g, 4.27 mmol, see international patentapplication WO 00/42059), PyBOP (1.89 g, 3.68 mmol), and DIPEA (1.06 g,8.23 mmol). The reaction was stirred at 0° C. for 2 h and then at roomtemperature overnight. The mixture was concentrated in vacuo and theresidue chromatographed two times on silica gel, eluting first withCHCl₃:EtOH (15:1) and second with EtOAc:EtOH (20:1) to afford the titlecompound (880 mg, 54%).

R_(f)=0.60 (10:1 CHCl₃:EtOH)

¹H NMR (300 MHz, CD₃OD, complex mixture of rotamers) δ 7.58-7.60 (d, J=8Hz, 2H), 7.34 (d, J=7 Hz, 2H), 7.05-7.08 (m, 2H), 6.95-6.99 (m, 1H),5.08-5.13 (m, 1H), 4.77-4.82 (m, 1H), 4.60-4.68 (m, 1H), 3.99-4.51 (m,7H), 3.82 (s, 3H), 2.10-2.75 (m, 2H).

¹³C-NMR (150 MHz; CD₃OD): (carbonyl and/or amidine carbons) δ 173.3,170.8, 152.5.

APCI-MS: (M+1)=493 m/z.

Preparation of Compound D (Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab)Compound D

Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab(Teoc) (0.045 g, 0.074 mmol; seePreparation A (ix) above), was dissolved in 3 mL of TFA and allowed toreact for 1 h. TFA was evaporated and the residue was freeze dried fromwater/acetonitrile to yield 0.043 g (100%) of the sub-title compound asits TFA salt.

¹H-NMR (400 MHz; CD₃OD) rotamers: δ 7.8-7.75 (m, 2H), 7.55-7.5 (m, 2H),7.35 (m, 1H, major rotamer), 7.31 (m, 1H, minor rotamer), 7.19 (m, 1H,major rotamer), 7.15 (m, 1H), 7.12 (m, 1H, minor rotamer), 6.89 (t, 1H,major rotamer), 6.87 (t, 1H, minor rotamer), 5.22 (m, 1H, minorrotamer), 5.20 (s, 1H, major rotamer), 5.13 (s, 1H, minor rotamer), 4.80(m, 1H, major rotamer), 4.6-4.4 (m, 2H), 4.37 (m, 1H, major rotamer),4.19 (m, 1H, major rotamer), 4.07 (m, 1H, minor rotamer), 3.98 (m, 1H,minor rotamer), 2.70 (m, 1H, minor rotamer), 2.55 (m, 1H, majorrotamer), 2.29 (m, 1H, major rotamer), 2.15 (m, 1H, minor rotamer)

¹³C-NMR (100 MHz; CD₃OD): (carbonyl and/or amidine carbons, rotamers) δ172.6, 172.5, 172.0, 171.7, 167.0

MS (m/z) 465 (M−1)⁻, 467 (M+1)⁺

Preparation of Compound E(Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab(2,6-diF)) Compound E

Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab(2,6-diF)(Teoc) (81 mg, 0.127mmol; see Preparation B (xii) above) was dissolved in 0.5 mL ofmethylene chloride and cooled on an ice bath. TFA (3 mL) was added andthe reaction was left for 75 min. The TFA was evaporated and the residuewas freeze dried from water and acetonitrile. The crude product waspurified by preparative RPLC with CH₃CN:0.1M NH₄OAc (35:65) to produce39 mg (55%) of the title compound as its HOAc salt, purity: 99%.

¹H NMR (400 MHz, CD₃OD mixture of rotamers) δ 7.5-7.4 (m, 2H), 7.32 (m,1H, major rotamer), 7.28 (m, 1H, minor rotamer), 7.2-7.1 (m, 3H) 6.90(t, 1H, major rotamer), 6.86 (t, minor rotamer), 5.15 (s, 1H, majorrotamer), 5.14 (m, 1H, minor rotamer), 5.07 (s, 1H, minor rotamer), 4.72(m, 1H, major rotamer), 4.65-4.45 (m, 2H), 4.30 (m, 1H, major rotamer),4.16 (m, 1H, major rotamer), 4.03 (m, 1H, minor rotamer), 3.95 (m, 1H,minor rotamer), 2.63 (m, 1H, minor rotamer), 2.48 (m, 1H, majorrotamer), 2.21 (m, 1H, major rotamer), 2.07 (m, 1H, minor rotamer), 1.89(s, 3H)

¹³C-NMR (75 MHz; CD₃OD): (carbonyl and/or amidine carbons, mixture ofrotamers) δ 171.9, 171.2, 165.0, 162.8, 160.4

APCI-MS: (M+1)=503/505 m/z.

Preparation of Compound F(Ph(3-Cl)(5-OCH₂CH₂F)—(R)CH(OH)C(O)-Aze-PabxTFA) (i)Ph(3-Cl)(5-OCH₂CH₂F)—(R)CH(OH)C(O)-Aze-Pab(Teoc)

To a solution of Ph(3-Cl)(5-OCH₂CH₂F)—(R)CH(OH)C(O)OH (940 mg, 3.78mmol; see Preparation C (v) above) in DMF (30 mL) under nitrogen at 0°C. was added HAze-Pab(Teoc)-HCl (2.21 g, 4.91 mmol), PyBOP (2.16 g, 4.15mmol), and DIPEA (1.22 g, 9.45 mmol). The reaction was stirred at 0° C.for 2 h and then at room temperature for 4 h. The mixture wasconcentrated in vacuo and the residue chromatographed twice on silicagel, eluting first with CHCl₃:EtOH (15:1) and second with EtOAc:EtOH(20:1) to afford the sub-title compound (450 mg, 20%) as a crushablewhite foam.

Mp: 80-88° C.

R_(f)=0.60 (10:1 CHCl₃:EtOH)

¹H NMR (300 MHz, CD₃OD, complex mixture of rotamers) δ 7.79 (d, J=8 Hz,2H), 7.42 (d, J=8 Hz, 2H), 7.05-7.08 (m, 1H), 6.93-6.99 (m, 2H),5.08-5.13 (m, 1H), 4.75-4.80 (m, 2H), 4.60-4.68 (m, 1H), 3.95-4.55 (m,8H), 2.10-2.75 (m, 2H), 1.05-1.11 (m, 2H), 0.08 (s, 9H).

APCI-MS: (M+1)=607 m/z.

(ii) Compound F

Ph(3-Cl)(5-OCH₂CH₂F)—(R)CH(OH)C(O)-Aze-Pab(Teoc) (0.357 g, 0.589 mmol;see step (i) above), was dissolved in 10 mL of TFA and allowed to reactfor 40 min. TFA was evaporated and the residue was freeze dried fromwater/acetonitrile to yield 0.33 g (93%) of the title compound as itsTFA salt.

¹H-NMR (600 MHz; CD₃OD) rotamers: δ 7.8-7.7 (m, 2H), 7.54 (d, 2H), 7.08(s, 1H, major rotamer), 7.04 (s, 1H, minor rotamer), 6.99 (s, 1H, majorrotamer), 6.95 (s, 1H), 6.92 (s, 1H, minor rotamer), 5.18 (m, 1H, minorrotamer), 5.14 (s, 1H, major rotamer), 5.08 (s, 1H, minor rotamer), 4.80(m, 1H, major rotamer), 4.73 (m, 1H), 4.65 (m, 1H), 4.6-4.4 (m, 2H),4.35 (m, 1H, major rotamer), 4.21 (doublet of multiplets, 2H), 4.12 (m,1H, major rotamer), 4.06 (m, 1H, minor rotamer), 3.99 (m, 1H, minorrotamer), 2.69 (m, 1H, minor rotamer), 2.53 (m, 1H, major rotamer), 2.29(m, 1H, major rotamer), 2.14 (m, 1H, minor rotamer).

¹³C-NMR (150 MHz; CD₃OD): (carbonyl and/or amidine carbons) δ 172.8,172.1, 167.4.

ESI-MS+: (M+1)=463 (m/z)

Preparation of Compound G (Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab(OH))(i) Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab(OH, Teoc)

Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab(Teoc) (0.148 g, 0.24 mmol; seePreparation A step (ix) above), was dissolved in 9 mL of acetonitrileand 0.101 g (1.45 mmol) of hydroxylamine hydrochloride was added. Themixture was heated at 70° C. for 2.5 h, filtered through Celite® andevaporated. The crude product (0.145 g; 75% pure) was used directly inthe next step without further purification.

(ii) Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab(OH)

Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab(OH, Teoc) (0.145 g, 0.23 mmol;see step (i) above), was dissolved in 0.5 mL of CH₂Cl₂ and 9 mL of TFA.The reaction was allowed to proceed for 60 minutes. TFA was evaporatedand the residue was purified using preparative HPLC. The fractions ofinterest were pooled and freeze-dried (2×), yielding 72 mg (yield overtwo steps 62%) of the title compound.

MS (m/z) 482 (M−1)⁻; 484 (M+1)⁺

¹H-NMR (400 MHz; CD₃OD): δ 7.58 (d, 2H), 7.33 (m, 3H), 7.15 (m, 2H),6.89 (t, 1H major rotamer), 6.86 (t, 1H minor rotamer), 5.18 (s, 1Hmajor rotamer; and m, 1H minor rotamer), 5.12 (s, 1H minor rotamer),4.77 (m, 1H major rotamer), 4.42 (m, 2H), 4.34 (m, 1H major rotamer),4.14 (m, 1H major rotamer), 4.06 (m, 1H minor rotamer), 3.95 (m, 1Hminor rotamer), 2.66 (m, 1H minor rotamer), 2.50 (m, 1H major rotamer),2.27 (m, 1H major rotamer), 2.14 (m, 1H minor rotamer)

¹³C-NMR (100 MHz; CD₃OD): (carbonyl and/or amidine carbons, rotamers) δ172.4, 172.3, 172.0, 171.4 152.3, 152.1

Preparation of Compound H:Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-duF)(OH)

(i) Boc-(S)Aze-NHCH₂-Ph(2,6-diF, 4-CN)

Boc-(S)Aze-OH (1.14 g, 5.6 mmol) was dissolved in 45 mL of DMF.4-Aminomethyl-2,6-difluorobenzonitrile (1.00 g, 5.95 mol, see Example1(xiv) above), PyBOP (3.10 g, 5.95 mmol) and DIPEA (3.95 mL, 22.7 mmol)were added and the solution was stirred at room temperature for 2 h. Thesolvent was evaporated and the residue was partitioned between H₂O andEtOAc (75 mL each). The aqueous phase was extracted with 2×50 mL EtOAcand the combined organic phase was washed with brine and dried overNa₂SO₄. Flash chromatography (SiO₂, EtOAc/heptane (3/1)) yielded thesub-title compound (1.52 g, 77%) as an oil which crystallized in therefrigerator.

¹H-NMR (400 MHz; CD₃OD): δ 7.19 (m, 2H), 4.65-4.5 (m, 3H), 3.86 (m, 1H),3.73 (m, 1H), 2.45-2.3 (m, 2H), 1.39 (s, 9H)

(ii) H—(S)Aze-NHCH₂-Ph(2,6-diF, 4-CN)xHCl

Boc-(S)Aze-NHCH₂-Ph(2,6-diF, 4-CN) (0.707 g, 2.01 mmol, see step (i)above) was dissolved in 60 mL of EtOAc saturated with HCl(g). Afterstirring at room temperature for 15 minutes, the solvent was evaporated.The residue was dissolved in CH₃CN/H₂O (1/1) and was freeze-dried togive the sub-title compound (0.567 g, 98%) as an off-white amorphouspowder.

¹H-NMR (400 MHz; CD₃OD): δ 7.49 (m, 2H), 4.99 (m, 1H), 4.58 (m, 2H),4.12 (m, 1H), 3.94 (m, 1H), 2.80 (m, 1H), 2.47 (m, 1H)

MS (m/z) 252.0 (M+1)⁺

(iii) Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-NHCH₂-Ph(2,6-diF, 4-CN)

Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)OH (0.40 g, 1.42 mmol, see Examplel(viii) above) was dissolved in 10 mL of DMF andH—(S)Aze-NHCH₂-Ph(2,6-diF, 4-CN)xHCl (0.43 g, 1.50 mmol, see step (ii)above) and PyBOP (0.779 g, 1.50 mmol) were added, followed by DIPEA (1.0mL, 5.7 mmol). After stirring at room temperature for 2 h, the solventwas evaporated. The residue was partitioned between H₂O (200 mL) andEtOAc (75 mL). The aqueous phase was extracted with 2×75 mL EtOAc andthe combined organic phase was washed with brine and dried over Na₂SO₄.Flash chromatography (SiO₂, EtOAc/heptane (4/1)) yielded the sub-titlecompound (0.56 g, 81%) as an oil.

¹H-NMR (400 MHz; CD₃OD) rotamers: δ 7.43 (m, 2H), 7.31 (m, 1H, majorrotamer), 7.26 (m, 1H, minor rotamer), 7.2-7.1 (m, 2H), 6.90 (t, 1H,major rotamer), 6.86 (t, 1H, minor rotamer), 5.14 (s, 1H, majorrotamer), 5.11 (m, 1H, minor rotamer), 5.04 (s, 1H, minor rotamer), 4.71(m, 1H, major rotamer), 4.6-4.45 (m, 2H), 4.30 (m, 1H, major rotamer),4.2-3.9 (m, 1H; and 1H, minor rotamer), 2.62 (m, 1H, minor rotamer),2.48 (m, 1H, major rotamer), 2.21 (m, 1H, major rotamer), 2.09 (m, 1H,minor rotamer)

¹³C-NMR (100 MHz; CD₃OD): (carbonyl carbons) δ 171.9, 171.8

MS (m/z) 484.0, 485.9 (M−1)⁻, 486.0, 487.9 (M+1)⁺

(iv) Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(OH)

Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-NHCH₂-Ph(2,6-diF, 4-CN) (0.555 g,1.14 mmol, from step (iii) above) was dissolved in 10 mL of EtOH (95%).To this solution was added hydroxylamine hydrochloride (0.238 g, 3.42mmol) and Et₃N (0.48 mL, 3.44 mmol). After stirring at room temperaturefor 14 h, the solvent was removed and the residue was dissolved inEtOAc. The organic phase was washed with brine and H₂O and was driedover Na₂SO₄. The crude product was purified by preparative RPLC withCH₃CN:0.1 M NH₄OAc as eluent, yielding the title compound as anamorphous powder (0.429 g, 72%) after freeze-drying.

¹H-NMR (400 MHz; CD₃OD) rotamers: δ 7.35-7.1 (m, 5H), 6.90 (t, 1H, majorrotamer), 6.85 (t, 1H, minor rotamer), 5.15 (s, 1H, major rotamer), 5.12(m, 1H, minor rotamer), 5.08 (s, 1H, minor rotamer), 4.72 (m, 1H, majorrotamer), 4.6-4.4 (m, 2H), 4.30 (m, 1H, major rotamer), 4.12 (m, 1H,major rotamer), 4.04 (m, 1H, minor rotamer), 3.94 (m, 1H, minorrotamer), 2.62 (m, 1H, minor rotamer), 2.48 (m, 1H, major rotamer), 2.22(m, 1H, major rotamer), 2.10 (m, 1H, minor rotamer)

¹³C-NMR (100 MHz; CD₃OD): (carbonyl and amidine carbons, rotamers) δ172.4, 171.9, 171.0, 152.3, 151.5

MS (m/z) 517.1, 519.0 (M−1)⁻, 519.1, 521.0 (M+1)⁺

Preparation of Compound J(Ph(3-Cl)(5-OCH₂CHF₂)—(R)CH(OH)C(O)-Aze-Pab(OH)) (i)Ph(3-Cl)(5-OCH₂CHF₂)—(R)CH(OH)C(O)-Aze-Pab(Z)

Boc-Aze-Pab(Z) (see international patent application WO 97/02284, 92 mg,0.197 mmol) was dissolved in 10 mL of EtOAc saturated with HCl(g) andallowed to react for 10 min. The solvent was evaporated and the residuewas mixed with Ph(3-Cl)(5-OCH₂CHF₂)—(R)CH(OH)C(O)OH (50 mg, 0.188 mmol;see Preparation C (v) above), PyBOP (109 mg, 0.209 mmol) and finallydiisopropylethyl amine (96 mg, 0.75 mmol) in 2 mL of DMF. The mixturewas stirred for 2 h and then poured into 50 mL of water and extractedthree times with EtOAc. The combined organic phase was washed withwater, dried (Na₂SO₄) and evaporated. The crude product was flashchromatographed on silica gel with EtOAc:MeOH (9:1). Yield: 100 mg(87%).

¹H NMR (300 MHz, CD₃OD, mixture of rotamers) δ 7.85-7.75 (m, 2H),7.45-7.25 (m, 7H), 7.11 (m, 1H, major rotamer), 7.08 (m, 1H, minorrotamer), 7.05-6.9 (m, 2H), 6.13 (bt, 1H), 5.25-5.05 (m, 3H), 4.77 (m,1H, partially hidden by the CD₃OH signal), 4.5-3.9 (m, 7H), 2.64 (m, 1H,minor rotamer), 2.47 (m, 1H, major rotamer), 2.25 (m, 1H, majorrotamer), 2.13 (m, 1H, minor rotamer)

(ii) Ph(3-Cl)(5-OCH₂CHF₂)—(R)CH(OH)C(O)-Aze-Pab(OH)

Hydroxylamine hydrochloride (65 mg, 0.94 mmol) and triethylamine (0.319g, 3.16 mmol) were mixed in 8 mL of THF and sonicated for 1 h at 40° C.Ph(3-Cl)(5-OCH₂CHF₂)—(R)CH(OH)C(O)-Aze-Pab(Z) (96 mg, 0.156 mmol; seestep (i) above) was added with 8 mL more of THF. The mixture was stirredat 40° C. for 4.5 days. The solvent was evaporated and the crude productwas purified by preparative RPLC with CH₃CN:0.1M NH₄OAc (40:60).

Yield: 30 mg (38%). Purity: 99%.

¹H NMR (300 MHz, CD₃OD, mixture of rotamers) δ 7.6-7.55 (m, 2H),7.35-7.3 (m, 2H), 7.12 (m, 1H, major rotamer), 7.09 (m, 1H, minorrotamer), 7.05-6.9 (m, 2H), 6.15 (triplet of multiplets, 1H), 5.15 (m,1H, minor rotamer), 5.13 (s, 1H, major rotamer), 5.08 (s, 1H, minorrotamer), 4.77 (m, 1H, major rotamer), 4.5-4.2 (m, 5H), 4.08 (m, 1H,major rotamer), 3.97 (m, 1H, minor rotamer), 2.66 (m, 1H, minorrotamer), 2.50 (m, 1H major rotamer), 2.27 (m, 1H, major rotamer), 2.14(m, 1H, minor rotamer).

¹³C-NMR (100 MHz; CD₃OD): (carbonyl and/or amidine carbons, mixture ofrotamers) δ 172.8, 172.2, 171.4, 159.1, 158.9, 154.2.

APCI-MS: (M+1)=497/499 m/z

Methods 1 and 2: Preparation of Salts of Compound A Method 1: GeneralMethod for Salt Preparation

The following generic method was employed to prepare salts of CompoundA: 200 mg of Compound A (see Preparation A above) was dissolved in 5 mLof MeOH. To this solution was added a solution of the relevant acid (1.0molar equivalent) dissolved in 5 mL of MeOH. After stirring for 10minutes at room temperature, the solvent was removed by way of a rotaryevaporator. The remaining solid material was re-dissolved in 8 mL ofacetonitrile:H₂O (1:1). Freeze-drying afforded colorless amorphousmaterial in each case.

Acids Employed:

-   (1S)-(+)-10-camphorsulfonic-   malic-   cyclohexylsulphamic-   phosphoric-   dimethylphosphoric-   p-toluenesulphonic-   L-lysine-   L-lysine hydrochloride-   saccharinic-   methanesulphonic-   hydrochloric

Appropriate characterising data are shown in Table 1. TABLE 1 δ ppm(MeOD) H18, H19, H24 Mw Mw (see structure at end Salt acid salt LRMS ofMethod 9 below) (1S)-(+)-10- 232.20 729.20 230.9 7.57, 7.68, 3.97camphorsulfonate 495.1 497.0 727.3 maleate 116.07 612.97 114.8 7.45,7.64, 3.89 495.1 497.0 cyclohexylsulphamate 179.24 676.14 177.9 7.44,7.64, 3.89 495.1 496.9 674.3 676.1 phosphate 97.99 594.89 495.1 7.37,7.61, 3.84 497.0 593.1 dimethylphosphate 126.05 622.95 124.9 7.50, 7.66,3.92 495.1 497.0 621.2 623.0 p-toluenesulphonate 172.20 669.10 170.97.54, 7.71, 3.95 495.1 497.0 L-lysine 146.19 643.09 145.0 7.36, 7.60,3.83 495.1 497.0 L-lysine hydrochloride 182.65 679.55 495.1 7.36, 7.60,3.83 497.0 531.1 (HCl) saccharinate 183.19 680.09 181.9 7.44, 7.64, 3.89495.1 497.0 methanesulphonate 96.11 593.01 495.1 7.57, 7.68, 3.97 497.0591.2 593.1 hydrochloride 36.46 533.36 495.1 7.55, 7.67, 3.95 496.9531.1 532.5 535.2

All salts formed in this Method were amorphous.

Method 2

Further amorphous salts of Compound A were made using analogoustechniques to those described in Method 1 above from the followingacids:

-   hydrobromic acid (1:1 salt)-   hydrochloric acid (1:1 salt)-   sulphuric acid (1:0.5 salt)-   1,2-ethanedisulfonic acid (1:0.5 salt)-   1S-camphorsulfonic acid (1:1 salt)-   (+/−)-camphorsulfonic acid (1:1 salt)-   ethanesulfonic acid (1:1 salt)-   nitric acid (1:1 salt)-   toluenesulfonic acid (1:1 salt)-   methanesulfonic acid (1:1 salt)-   p-xylenesulfonic acid (1:1 salt)-   2-mesitylenesulfonic acid (1:1 salt)-   1,5-naphthalenesulfonic acid (1:0.5 salt)-   naphthalenesulfonic acid (1:1 salt)-   benzenesulfonic acid (1:1 salt)-   saccharinic acid (1:1 salt)-   maleic acid (1:1 salt)-   phosphoric acid (1:1 salt)-   D-glutamic acid (1:1 salt)-   L-glutamic acid (1:1 salt)-   D,L-glutamic acid (1:1 salt)-   L-arginine (1:1 salt)-   L-lysine (1:1 salt)-   L-lysine hydrochloride (1:1 salt)-   glycine (1:1 salt)-   salicylic acid (1:1 salt)-   tartaric acid (1:1 salt)-   fumaric acid (1:1 salt)-   citric acid (1:1 salt)-   L-(−)-malic acid (1:1 salt)-   D,L-malic acid (1:1 salt)-   D-gluconic acid (1:1 salt)

Method 3: Preparation of Amorphous Compound A, ethanesulfonic acid salt

Compound A (203 mg; see Preparation A above) was dissolved in ethanol (3mL) and ethanesulfonic acid (1 eq., 95%, 35 μL) was added to thesolution. The mixture was stirred for a few minutes, and then thesolvent was evaporated. The resulting oil was slurried in iso-octane andevaporated to dryness until a solid material was obtained. Finally, thesubstance was re-slurried in iso-octane and the solvent evaporated againresulting in a white, dry, amorphous solid. The substance was vacuumdried at 40° C. overnight.

Methods 4 to 9: Preparation of Crystalline Compound A, ethanesulfonicacid salt Method 4: Crystallisation of Amorphous Material

Amorphous Compound A, ethanesulfonic acid salt (17.8 mg; see Method 3above) was slurried in methyl iso-butyl ketone (600 μL). After 1 week,crystalline needles were observed, which were filtered off andair-dried.

Methods 5 to 7: Reaction Crystallisations (without Anti-solvent) Method5

Compound A (277 mg; see Preparation A above) was dissolved in methyliso-butyl ketone (3.1 mL). Ethanesulfonic acid was added (1 eq., 95%, 48μL). Precipitation of amorphous ethanesulfonate salt occurredimmediately. More methyl iso-butyl ketone (6 mL) was added and theslurry was treated with ultrasound. Finally, a third portion of methyliso-butyl ketone (3.6 mL) was added and then the slurry was leftovernight with stirring (magnetic stirrer). The next day, the substancehad transformed into crystalline needles. The slurry was filtered off,washed with methyl iso-butyl ketone (0.5 mL) and air dried.

Method 6

Compound A (236 mg; see Preparation A above) was dissolved at roomtemperature in methyl iso-butyl ketone (7 mL). Ethanesulfonic acid (1eq., 41 μL) was mixed with 2 mL of methyl iso-butyl ketone in a vial.The solution of Compound A was seeded with crystalline Compound A,ethanesulfonic acid salt (see Methods 4 and 5 above). Then, 250 μL ofthe methyl iso-butyl ketone solution of ethanesulfonic acid was added inportions over 45 minutes. The solution was seeded again, and thetemperature was increased to 30° C. Then, 500 μL of the methyl iso-butylketone solution was added over approximately 1 hour. The resultingslurry was left overnight before a final amount of the methyl iso-butylketone/acid solution was added over 20 minutes. The vial was rinsed with1.5 mL of methyl iso-butyl ketone, which was added to the slurry. Aftera further 6 hours, the crystals were filtered off, washed with methyliso-butyl ketone (2 mL) and dried under reduced pressure at 40° C. Atotal of 258 mg of crystalline salt was obtained which corresponds to ayield of approximately 87%.

Method 7

Compound A (2.36 g; see Preparation A above) was dissolved in methyliso-butyl ketone (90 mL). Seed crystals (10 mg) of Compound A,ethanesulfonic acid salt (see Methods 4 to 6 above) were added to thesolution, and then ethanesulfonic acid (40 μL) was added in twoportions. Further seed crystals (12 mg) and two portions ofethanesulfonic acid (2×20 μL) were then added. The slurry was dilutedwith methyl iso-butyl ketone (15 mL) before the addition ofethanesulfonic acid was continued. A total amount of 330 μLethanesulfonic acid was added, in portions, over 1 hour. A small amountof seed crystals was added and, finally, the slurry was left overnightwith stirring. The next day, the crystals were filtered off, washed withmethyl iso-butyl ketone (2×6 mL) and dried under reduced pressure at 40°C. After drying, a total of 2.57 g of white, crystalline product wasobtained corresponding to a yield of 89%.

Methods 8 and 9: Reaction Crystallizations (with Anti-Solvent) Method 8

Compound A (163 mg; see Preparation A above) was dissolved iniso-propanol (1.2 mL). The solution was heated to 35° C. Ethanesulfonicacid was added (28 μL). Then, ethyl acetate (4.8 mL) was added and thesolution was seeded with crystalline Compound A, ethanesulphonic acidsalt (see Methods 4 to 7 above). Crystallization started almostimmediately. The slurry was left for about 80 minutes at 35° C. beforebeing allowed to cool to ambient temperature (21° C.). Two hours later,the crystals were filtered off, washed three times with ethyl acetate(3×0.4 mL), and dried under reduced pressure at 40° C. A total of 170 mgof crystalline title product was obtained which corresponds to a yieldof approximately 82%.

Method 9

Compound A (20.0 g; see Preparation A above) was dissolved iniso-propanol (146.6 mL) at 40° C. and ethanesulfonic acid (3.46 mL, 95%,1 eq.) was added to the solution. To the resulting clear solution, seedcrystals of Compound A, ethanesulfonic acid salt were added (50 mg; seeMethods 4 to 8 above). Then, ethyl acetate (234 mL) was added over 10minutes. The resulting slightly opaque solution was seeded once more (70mg) and left for one hour at 40° C. with stirring to allow forcrystallization to start. After this, a total of 352 mL of ethyl acetatewas added at a constant rate over one hour. When all of the ethylacetate had been added, the slurry was left for 1 hour, before beingcooled to 21° C. over 2 hours. The crystallization was allowed tocontinue for 1 hour at 21° C. before the crystals were filtered off,washed twice with ethyl acetate (50 mL+60 mL) and finally, dried underreduced pressure at 40° C. overnight. A total of 21.6 g of a white,crystalline salt was obtained, corresponding to a yield of approximately90%.

Compound A, ethanesulfonic acid salt was characterised by NMR asfollows: 23 mg of the salt was dissolved in deuterated methanol (0.7 mL)troscopy. A combination of 1D (¹H, ¹³C and selective NOE) and 2D (gCOSY,gHSQC and gHMBC) NMR experiments were used. All data were in goodagreement with the theoretical structure of the salt, shown below. Themolecule exists in two conformations in methanol. Based on the integralof the peak assigned to H5 (dominant conformer) and peak assigned to H5′(other conformer), the ratio between the two conformers was found to be70:30. H22 could not be observed as these protons were in fast exchangewith the solvent CD₃OD.

Both the proton and the carbon resonance corresponding to position 1 aresplit due to the spin-coupling with the two fluorine nuclei in thatposition. The coupling constants are ²J_(HF)=73 Hz and ¹J_(CF)=263 Hz.

¹H and ¹³C NMR chemical shift assignment and proton-proton correlationsare shown in Table 2. TABLE 2 Atom ¹³C shift/ ¹H shift/ppm^(b) and No.Type ppm^(a) multiplicity^(c) J_(HH)/Hz  1 CH 117.5^(e) 6.90 (t) 73(²J_(HF))   1′ 117.5^(e) 6.88 (t)  2 C 153.5   2′ 153.5  3 CH 120.0 7.15(s)   3′ 119.7 7.13 (s)  4 C 136.2   4′ 135.9  5 CH 125.0 7.36 (s)   5′124.9 7.31 (s)  6 C 144.5   6′ 145.3  7 CH 117.3 7.20 (s)   7′ 117.27.15 (s)  8 CH 72.0 5.20 (s)   8′ 74.0 5.12 (s)  9 CO 173.1   9′ 173.811 CH₂ 51.6 a: 4.38 (m) b: 4.21 (m)  11′ 49.0 a: 4.06 (m) b: 3.99 (m) 12CH₂ 21.7 a: 2.55 (m) b: 2.29 (m)  12′ 23.2 a: 2.70 (m) b: 2.15 (m) 13 CH63.1 4.80 (m)  13′ 66.2 5.22 (m) 14 CO 172.9  14′ 173.6 15 NH 8.76 (t,br) 5.2  15′ 8.79 (t, br) 5.2 16 CH₂ 43.5 4.59 (AB-pattern) 15.9 4.46(AB-pattern) 15.9  16′ 43.6 4.53 (AB-pattern) 15.9 4.49 (AB-pattern)15.9 17 C 146.9  17′ 147.0 18 CH 129.1 7.56 (d) 7.8  18′ 129.1 7.57 (d)7.8 19 CH 129.2 7.67 (d) 7.8  19′ 129.4 7.70 (d) 7.8 20 C 124.9 —  20′124.9 21 C 162.4  21′ 162.3 22 NH₂ Not observed 24 CH₃ 64.8 3.96 (s)101  CH3 1.28 (t) 7.4 102  CH2 2.77 (m) 7.4^(a)Relative to the solvent resonance at 49.0 ppm.^(b)Relative to the solvent resonance at 3.30 ppm.^(c)s = singlet, t = triplet, m = multiplet, br = broad, d = doublet^(d)Obtained in the gCOSY experiment.^(e)The resonance is a triplet due to coupling with the two fluorinenuclei.¹J_(CF) = 263 Hz.

HRMS calculated for C₂₄H₂₉ClF₂N₄O₈S (M−H)hu − 605.1284, found 605.1296.

Crystals of Compound A, ethanesulfonic acid salt (obtained by way of oneor more of Examples 4 to 9 above) were analyzed by XPAD and the resultsare tabulated below (Table 3) and are shown in FIG. 1. TABLE 3 d value(Å) Intensity (%) Intensity 16.5 10 m 12.2 74 vs 11.0 4 w 9.0 33 s 8.3 3vw 7.6 6 w 6.4 4 w 6.2 12 m 6.0 7 m 5.9 10 m 5.5 15 m 5.4 100 vs 5.1 7 m4.66 29 s 4.60 36 s 4.31 57 s 4.25 18 m 4.19 20 m 4.13 12 m 4.00 12 m3.87 13 m 3.83 6 w 3.76 7 m 3.72 6 w 3.57 9 m 3.51 7 m 3.47 5 w 3.39 3vw 3.31 11 m 3.26 10 m 3.21 8 m 3.16 4 w 3.03 8 m 2.78 4 w 2.74 5 w 2.673 vw 2.56 5 w 2.50 5 w 2.46 7 m 2.34 4 w 2.21 5 w 2.00 3 vw 1.98 3 vw

DSC showed an endotherm with an extrapolated melting onset temperatureof ca. 131° C. TGA showed a decrease in mass of ca. 0.2% (w/w) aroundthe melting point. DSC analysis repeated with a sample of lower solventcontent showed a melting onset temperature of ca. 144° C.

Method 10: Preparation of Amorphous Compound A, benzenesulfonic acidsalt

Compound A (199 mg; see Preparation A above) was dissolved in ethanol (2mL). Benzenesulfonic acid (1 eq. 90%, 70 mg) was dissolved in ethanol (1mL) in a vial. The ethanol solution of the acid was added to thesolution of Compound A and the vial was rinsed with 1 mL ethanol, whichwas then added to the mixture. The mixture was stirred for a fewminutes, and then the ethanol was evaporated until an oil was formed.Ethyl acetate (3 mL) was added and the solvent was evaporated again todryness. An amorphous solid was formed.

Methods 11 to 13: Preparation of Crystalline Compound A, benzenesulfonicacid salt Method 11: Crystallisation of Amorphous Material

Amorphous Compound A benzenesulfonic acid salt (20.7 mg; see Method 10above) was slurried in ethyl acetate (600 TL). After 5 days, crystallineneedles were observed in the slurry.

Methods 12 and 13: Reaction Crystallisations Method 12

Compound A (128 mg; see Preparation A above) was dissolved in ethylacetate (3 mL). The solution was seeded with the slurry from Method 11above. Then, benzenesulfonic acid was added (1 eq., 90%, 45 mg).Precipitation of benzenesulphonic acid salt occurred immediately.iso-Propanol was added to the slurry (0.8 mL) and the mixture was seededagain. Two days later, the substance had transformed into crystallineneedles. The slurry was filtered off, washed with ethyl acetate (3×0.2mL) and dried for a short time under vacuum at 40° C. A total ofapproximately 140 mg of white solid was obtained.

Method 13

Compound A (246 mg; see Preparation A above) was dissolved iniso-propanol (1.52 mL). Benzenesulfonic acid was added (88 mg, 90%). Tothe clear solution, ethyl acetate was added (3 mL), and then the mixturewas seeded to initiate crystallisation. After 1 hour, more ethyl acetatewas added (2.77 mL). Finally, the slurry was allowed to crystalliseovernight before the crystals were filtered off, washed with ethylacetate (3×0.3 mL) and dried at 40° C. under vacuum. A total of 279 mgsalt was obtained which corresponds to a yield of approximately 86%.

Compound A, benzenesulfonic acid salt was characterised by NMR asfollows: 20 mg of the salt was dissolved in deuterated methanol (0.7mL). A combination of 1D (¹H, ¹³C and selective NOE) and 2D (gCOSY,gHSQC and gHMBC) NMR experiments were used. All data were in goodagreement with the theoretical structure of the salt, shown below. Themolecule exists in two conformations in methanol. Based on the integralof the peak assigned to H12 (dominant conformer) and peak assigned toH12′ (other conformer), the ratio between the two conformers was foundto be 70:30. H22 could not be observed as these protons were in fastexchange with the solvent CD₃OD.

Both the proton and the carbon resonance corresponding to position 1 aresplit due to the spin-coupling with the two fluorine nuclei in thatposition. The coupling constants are ²J_(HF)=74 Hz and ¹J_(CF)=260 Hz.

¹H and ¹³C NMR chemical shift assignment and proton-proton correlationsare shown in Table 4. TABLE 4 Atom ¹H shift/ppm^(b) and No. Type ¹³Cshift/ppm^(a) multiplicity^(c) J_(HH)/Hz  1 CH 117.5^(e) 6.89 (t) 74(²J_(HF))   1′ 117.5^(e) 6.87 (t)  2 C 153.5   2′ 153.5  3 CH 120.1 7.15(s)   3′ 119.7 7.12 (s)  4 C 136.2   4′ 135.9  5 CH 125.1 7.35 (s)   5′124.9 7.31 (s)  6 C 144.5   6′ 145.3  7 CH 117.3 7.20 (s)   7′ 117.27.14 (s)  8 CH 72.8 5.20 (s)   8′ 74.0 5.12 (s)  9 CO 173.1   9′ 173.811 CH₂ 51.6 a: 4.37 (m) b: 4.20 (m)  11′ 49.0 a: 4.05 (m) b: 3.98 (m) 12CH₂ 21.7 a: 2.53 (m) b: 2.28 (m)  12′ 23.2 a: 2.69 (m) b: 2.14 (m) 13 CH63.1 4.79 (m)  13′ 66.2 5.22 (m) 14 CO 172.9  14′ 173.6 15 NH 8.75 (t,br) 5.3  15′ 8.78 (t, br) 5.3 16 CH₂ 43.5 4.59 (AB-pattern) 16.0 and 5.24.44 (AB-pattern) 16.0 and 4.8  16′ 43.6 4.51 (AB-pattern) 16.0  4.46(AB-pattern) 16.0  17 C 146.9  17′ 147.0 18 CH 129.2 7.54 (d) 8.3  18′129.2 7.56 (d) 8.3 19 CH 129.3 7.66 (d) 8.3  19′ 129.4 7.69 (d) 8.3 20 C124.9 —  20′ 124.9 21 C 162.4  21′ 162.4 22 NH₂ Not observed 24 CH₃ 64.83.95 (s) 101  CH 126.9 7.81 (m) 102  CH 129.1 7.41 (m) 103  CH 131.27.42 (m) 104  C 146.4^(a)Relative to the solvent resonance at 49.0 ppm.^(b)Relative to the solvent resonance at 3.30 ppm.^(c)s = singlet, t = triplet, m = multiplet, br = broad, d = doublet.^(d)Obtained in the gCOSY experiment.^(e)The resonance is a triplet due to coupling with the two fluorinenuclei.¹J_(CF) = 260 Hz.^(f)connectivity difficult to determine due to overlap between resonance102 and 103

HRMS calculated for C₂₈H₂₉ClF₂N₄O₈S (M−H)⁻ 653.1284, found 653.1312.

Crystals of Compound A, benzenesulfonic acid salt (obtained by way ofone or more of Examples 11 to 13 above) were analyzed by XRPD and theresults are tabulated below (Table 5) and are shown in FIG. 2. TABLE 5 dvalue (Å) Intensity (%) Intensity 14.2 12 m 12.6 55 s 10.2 49 s 7.5 8 m6.4 5 w 6.3 30 s 6.1 5 w 5.9 100 vs 5.7 20 m 5.4 9 m 5.3 11 m 5.1 10 m4.96 3 vw 4.83 27 s 4.73 72 vs 4.54 23 s 4.50 10 m 4.35 28 s 4.30 38 s4.24 24 s 4.17 28 s 4.09 60 vs 4.08 61 vs 3.96 29 s 3.91 15 m 3.77 22 s3.62 11 m 3.52 20 m 3.31 44 s 3.19 8 m 3.15 11 m 3.09 8 m 3.00 7 m 2.893 vw 2.86 4 w 2.79 7 m 2.76 6 w 2.72 5 w 2.59 6 w 2.56 9 m 2.54 9 m 2.497 m 2.38 8 m 2.16 4 w 2.03 3 vw

DSC showed an endotherm with an extrapolated melting onset temperatureof ca. 152° C. TGA showed a decrease in mass of ca. 0.1% (w/w) aroundthe melting point.

Method 14: Preparation of Amorphous Compound A, n-propanesulfonic acidsalt

Compound A (186 mg; see Preparation A above) was dissolved iniso-propanol (1.39 mL) and n-propanesulfonic acid (1 eq., 95%, 39 TL)was added. Ethyl acetate (5.6 mL) was added and the solvent wasevaporated until a dry, amorphous solid was formed.

Methods 15 and 16: Preparation of Crystalline Compound A,n-propanesulfonic acid salt Method 15: Crystallisation of AmorphousMaterial

Amorphous Compound A, n-propanesulfonic acid salt (20 mg; see Method 14above) was dissolved in iso-propanol (60 TL) and iso-propyl acetate (180TL) was added. After three days crystalline needles were observed.

Method 16: Reaction Crystallisation

Compound A (229 mg; see Preparation A above) was dissolved iniso-propanol (1.43 mL). n-Propanesulfonic acid was added (1 eq., 95%, 48TL). Ethyl acetate was added (2 mL), and then the solution was seededwith crystalline salt from Method 15 above. Further ethyl acetate wasadded (5 mL) and the slurry was left overnight to crystallize. Thecrystals were filtered off, washed with ethyl acetate (3×0.3 mL) anddried under vacuum at 40° C.

Compound A, n-propanesulfonic acid salt was characterised by NMR asfollows: 13 mg of the salt was dissolved in deuterated methanol (0.7 mL)troscopy. A combination of 1D (¹H, ¹³C) and 2D (gCOSY) NMR experimentswere used. All data were in good agreement with the theoreticalstructure of the salt, shown below. The molecule exists in twoconformations in methanol. Based on the integral of the peak assigned toH12 (dominant conformer) and peak assigned to H12′ (other conformer),the ratio between the two conformers was found to be 65:35. H22 couldnot be observed as these protons were in fast exchange with the solvent

Both the proton and the carbon resonance corresponding to position 1 aresplit due to the spin-coupling with the two fluorine nuclei in thatposition. The coupling constants are ²J_(HF)=74 Hz and ¹J_(CF)=260 HZ.

¹H and ¹³C NMR chemical shift assignment and proton-proton correlationsare shown in Table 6. TABLE 6 Atom ¹H shift/ppm^(b) and No. Type ¹³Cshift/ppm^(a) multiplicity^(c) J_(HH)/Hz  1 CH 117.5^(e) 6.89 (t) 74(²J_(HF))   1′ 117.5^(e) 6.88 (t)  2 C 153.5   2′ 153.5  3 CH 120.0 7.16(s)   3′ 119.7 7.13 (s)  4 C 136.2   4′ 135.9  5 CH 125.1 7.36 (s)   5′124.9 7.31 (s)  6 C 144.5   6′ 145.3  7 CH 117.3 7.20 (s)   7′ 117.27.16 (s)  8 CH 72.9 5.20 (s)   8′ 74.1 5.12 (s)  9 CO 173.1   9′ 173.811 CH₂ 51.6 a: 4.37 (m) b: 4.20 (m)  11′ 49.0 a: 4.06 (m) b: 3.98 (m) 12CH₂ 21.7 a: 2.53 (m) b: 2.29 (m)  12′ 23.2 a: 2.69 (m) b: 2.15 (m) 13 CH63.1 4.80 (m)  13′ 66.2 5.22 (m) 14 CO 172.9  14′ 173.8 15 NH 8.75 (t,br) 5.5  15′ 8.79 (t, br) 5.5 16 CH₂ 43.5 4.59 (AB-pattern) 16.0 and 6.64.45 (AB-pattern) 16.0 and 5.3  16′ 43.6 4.51 4.50 17 C 146.9  17′ 147.018 CH 129.1 7.54 (d) 8.5  18′ 129.2 7.57 (d) 8.5 19 CH 129.2 7.67 (d)8.5  19′ 129.4 7.69 (d) 8.5 20 C 124.9 —  20′ 124.9 21 C 162.4  21′162.4 22 NH₂ Not observed 24 CH₃ 64.7 3.96 (s) 101  CH 13.7 1.0 (t) 102 CH 19.6 1.78 (m) 103  CH 54.6 2.75 (m)^(a)Relative to the solvent resonance at 49.0 ppm.^(b)Relative to the solvent resonance at 3.30 ppm.^(c)s = singlet, t = triplet, m = multiplet, br = broad, d = doublet.^(d)Obtained in the gCOSY experiment.^(e)The resonance is a triplet due to coupling with the two fluorinenuclei.¹J_(CF) = 260 Hz.

HRMS calculated for C₂₅H₃₁ClF₂N₄O₈S (M−H)⁻ 619.1441, found 619.1436.

Crystals of Compound A, n-propanesulfonic acid salt (obtained by way ofone or more of Examples 15 and 16 above) were analyzed by XRPD and theresults are tabulated below (Table 7) and are shown in FIG. 3. TABLE 7 dvalue (Å) Intensity (%) Intensity 14.0 4 w 12.4 87 vs 10.0 30 s 8.0 3 vw7.5 7 m 7.0 0.6 vw 6.7 1 vw 6.4 1 vw 6.2 12 m 6.1 3 vw 5.8 100 vs 5.7 11m 5.5 3 vw 5.4 5 w 5.3 5 w 5.2 2 vw 5.1 3 vw 4.94 3 vw 4.78 21 s 4.68 42s 4.51 10 m 4.49 7 m 4.40 5 w 4.32 10 m 4.29 10 m 4.25 22 s 4.19 14 m4.14 15 m 4.07 23 s 4.04 20 m 3.94 16 m 3.88 10 m 3.73 15 m 3.65 2 vw3.59 3 vw 3.48 18 m 3.28 23 m 3.12 4 w 3.06 3 vw 2.97 6 w 2.84 2 vw 2.813 vw 2.76 2 vw 2.73 3 vw 2.70 2 vw 2.57 2 vw 2.54 6 w 2.51 6 w 2.46 8 m2.42 2 vw 2.39 3 vw 2.36 3 vw 2.32 2 vw 2.14 3 vw 2.01 2 vw

DSC showed an endotherm with an extrapolated melting onset temperatureof ca. 135° C. TGA showed no decrease in mass around the melting point.

Method 17 Method 17-A: Preparation of amorphous Compound A n-butanesulfonic acid salt

Amorphous Compound A (277 mg) was dissolved in IPA (1.77 ml) and butanesulfonic acid (approx. 1 eq. 70 μL) was added. Ethyl acetate (6 ml) wasadded and the solvent was evaporated until dry, amorphous solid wasformed.

Method 17-B: Preparation of Crystalline Compound A Butane sulfonic acidsalt

Amorphous Compound A butane sulfonic acid salt (71.5 mg; see preparationabove) was slurried in ethyl acetate (500 μl) over night. The crystalswere filtered off and were air-dried.

Compound A, butanesulfonic acid salt was charaterised by NMR as follows:

21.6 mg of the salt was dissolved in deuterated dimethylsulfoxide (0.7ml) and was investigated with ¹H and ¹³C NMR spectroscopy.

The spectra are very similar to other salts of the same compound and ingood agreement with the structure shown below. Most resonances in thespectra are present as sets of two peaks due to the slow rotation aroundthe C9-N10 bond, which results in two atropisomers that simultaneouslyexist in the solution. This is shown for other salts of the samecompound.

The two fluorine nuclei in position 1 give rise to split resonances forthe proton and the carbon in that position. The coupling constants are²J_(HF)=73 Hz and ¹J_(CF)=258 Hz. Chemical shifts for protons andcarbons are presented in Table 1. Protons in position 22 and 24 are notdetected due to chemical exchange. There is a very broad hump between 8and 9 ppm in the proton spectrum corresponding to these protons. TABLE 8¹H and ¹³C NMR chemical shift assignment of Compound A n-butanesulfonatesalt in deuterated dimethylsulfoxide at 25° C. Atom ¹³C shift/ ¹Hshift/ppm^(b) and No. Typ ppm^(a) multiplicity^(c) J_(HH)/Hz  1 CHF₂116.3^(d) 7.29 (t) 73 (²J_(HF))   1′ 116.3^(d) 7.28 (t) 73 (²J_(HF))  2C 151.5 na na   2′ 151.3 na na  3 CH 118.0 7.25 (t)^(e) nd   3′ 117.67.21 (t)^(e) nd  4 C 133.8 na na   4′ 133.4 na na  5 CH 123.8 7.34(t)^(e) nd   5′ 123.6 7.25 (t)^(e) nd  6 C 144.5 na na   6′ 145.2 na na 7 CH 116.3 7.19 (t)^(e) nd   7′ 116.1 7.12 (t)^(e) nd  8 CH 70.9 5.13(s) na   8′ 71.2 4.99 (s) na  9 CO 170.6 na na   9′ 171.1 na na 11 CH₂50.0 a: 4.24 (m) b: 4.12 (m) nd  11′ 46.9 3.85 (m) nd 12 CH₂ 20.5 a:2.41 (m) b: 2.10 (m) nd  12′ 21.7 a: 2.60 (m) b: 2.02 (m) nd 13 CH 61.24.65 (dd) 5.6 and 8.9  13′ 63.9 5.12 (m) nd 14 CO 170.2 na na  14′ 171.0na na 16 CH₂ 41.8 4.38 (m) nd  16′ 42.0 4.38 (m) nd 17 C 144.7 na na 18CH 127.5 7.44 (d) 8.2 127.6 7.44 nd 19 CH 127.8 7.66 (d) 8.2 20 C 125.1na na 21 C 157.9 na na 24 CH₃ 63.3 3.83 (s) na  24′ 63.3 3.82 (s) na 26CH₂ 51.4 2.41 (m) nd 27 CH₂ 27.3 1.52 (m) nd 28 CH₂ 21.7 1.30 (m) nd 29CH₃ 14.0 0.83 (t) 7.3^(a)Relative to the solvent resonance at 49.0 ppm.^(b)Relative to the solvent resonance at 3.30 ppm.^(c)s = singlet, d = doublet, dd = doublet of doublets, t = triplet, m =multiplet.^(d)The resonance is a triplet due to coupling with the two fluorinenuclei F1.¹J_(CF) = 258 Hz.^(e)The ⁴J_(HH) coupling with the meta-protons is not fully resolved.na = not applicable,nd = not determined

HRMS calculated for C₂₆H₃₂ClF₂N₄O₈S (M−H)⁻ 633.1597, found 633.1600

Crystals of Compound A n-butanesulfonic acid salt (obtained as describedabove in Method 17-B) were analyzed by XRPD and the results aretabulated below (Table 9) and are shown in FIG. 4. TABLE 9 d-value (Å)Intensity (%) Intensity 14.3 8 m 12.8 81 vs 10.3 44 s 8.2 4 w 7.7 13 m6.7 2 vw 6.4 8 m 6.2 18 m 6.0 100 vs 5.8 29 s 5.6 4 w 5.4 11 m 5.3 16 m5.1 15 m 4.98 6.5 w 4.91 34 s 4.76 56 s 4.57 20 m 4.42 13 m 4.36 19 m4.30 45 s 4.18 42 s 4.13 88 vs 4.01 34 s 3.92 28 s 3.82 18 m 3.64 6.6 w3.58 16 m 3.47 5 w 3.44 6 w 3.38 12 m 3.35 32 s 3.32 22 s 3.29 12 m 3.208 m 3.17 9 m 3.02 12 m 2.90 6 w 2.81 3.9 vw 2.75 3 vw 2.64 3.5 vw 2.5910 m 2.57 8 m 2.50 4 w 2.45 5 w 2.40 6 w 2.31 3 vw

DSC showed an endotherm with an extrapolated melting onset temperatureof ca 118° C. and TGA showed a 0.04% weight loss.

Method 18: Preparation of Salts of Compound B Method 18-A: GeneralMethod for Salt Preparation

The following generic method was employed to prepare salts of CompoundB: 200 mg of compound B (see Preparation B above) was dissolved in 5 mLof MIBK (methyl isobutyl ketone). To this solution was added a solutionof the relevant acid (1.0 or 0.5 molar equivalent, as indicated in Table10) dissolved in 1.0 mL of MIBK. After stirring for 10 minutes at roomtemperature, the solvent was removed by way of a rotary evaporator. Theremaining solid material was re-dissolved in about 8 mL ofacetonitrile:H₂O (1:1). Freeze-drying afforded colorless amorphousmaterial in each case.

Acid Employed:

-   Esylate (ethanesulfonic acid)-   Besylate (benzene sulfonic acid)-   Cyclohexylsulphamate-   Sulphate-   Bromide-   p-Toluenesulphonate-   2-Naphtalenesulfonate-   Hemisulfate-   Methanesulphonate-   Nitrate-   Hydrochloride

Appropriate characterising data are shown in Table 10 TABLE 10 Salt Mwacid Mw salt MS ES- Esylate 110.13 643.01 108.8 531.1 641.0 Besylate158.18 691.06 156.8 531.1 689.2 Cyclohexylsulphamate 179.24 712.12 177.9531.2 710.4 Sulphate 98.08 630.96 531.1 Bromide 80.91 613.79 531.2 613.1p-Toluenesulphonate 172.20 705.08 170.9 531.1 703.1 2- 208.24 741.12206.9 Naphtalenesulfonate 531.1 739.3 Hemisulfate 98.07 1163.8  531.1(1:2) 631.0 630.85 (1:1) Methanesulphonate 96.11 628.99 531.1 627.1Nitrate 63.01 595.89 531.0 594.0 Hydrochloride 36.46 569.34 531.0 569.0

All salts formed in this Example were amorphous.

Method 18-B

Further amorphous salts of Compound B were made using analogoustechniques to those described in Method 18-A above for the followingacids:

-   1,2-Ethanedisulfonic (0.5 salt)-   1S-Camphorsulfonic-   (+/−)-Camphorsulfonic-   p-Xylenesulfonic-   2-Mesitylenesulfonic-   Saccharin-   Maleic-   Phosphoric-   D-glutamic-   L-arginine-   L-lysine-   L-lysine*HCl

Method 18-C: Preparation of Amorphous Compound B,Hemi-1,5-naphtalenedisulfonic acid salt

Amorphous Compound B (110.9 mg) was dissolved in 2.5 mL 2-propanol and0.5 equivalent of 1,5-naphthalene-disulfonic acid tetrahydrate was added(dissolved in 1 mL 2-propanol). The sample was stirred overnight. Onlysmall particles (amorphous) or oil drops were observed by microscopy.The sample was evaporated to dryness.

Method 18-D: Preparation of Crystalline Compound B,hemi-1,5-naphtalenedisulfonic acid salt

The crystallization experiment was carried out at ambient temperature.Amorphous Compound B (0.4 gram) was dissolved in ethanol (1.5 mL) and0.5 eq of 1,5-naphthalene-disulfonic acid tetrahydrate (1.35 gram, 10%in ethanol) was added. Heptane (0.7 mL) was then added until thesolution became slightly cloudy. After about 15 minutes the solutionbecame turbid. After about 30 minutes thin slurry was obtained andadditional heptane (1.3 mL) was added. The slurry was than leftovernight for ripening. To dilute the thick slurry, a mixture of ethanoland heptane (1.5 mL and 1.0 mL respectively) was added. After about 1hour the slurry was filtered and the crystals were washed with a mixtureof ethanol and heptane (1.5:1) and finally with pure heptane. Thecrystals were dried at ambient temperature in 1 day. The dry crystalsweighed 0.395 g.

Method 18-E: Preparation of Crystalline Compound B,hemi-1,5-naphtalenedisulfonic acid salt

Amorphous Compound B (1.009 gr) was dissolved in 20 mL 2-propanol+20 mLethyl acetate. 351.7 mg 1,5-naphtalene-disulfonic acid tetrahydrate,dissolved in 20 mL 2-propanol, was added drop by drop. Precipitationoccurred in about 5 minutes. The slurry was stirred over night and thenfiltered.

Method 18-F: Preparation of Crystalline Compound B,hemi-1,5-naphtalenedisulfonic acid salt

430.7 mg of the 1,5-naphtalene-disulfonic acid salt was dissolved in 30mL 1-propanol. The solution was heated to boiling in order to dissolvethe substance. The solution was left over night at ambient temperaturefor crystallization and then the crystals were filtered off.

Method 18-G: Preparation of Crystalline Compound B,hemi-1,5-naphtalenedisulfonic acid salt

The mother liquid from Method 18-F was evaporated and the solid rest(61.2 mg) was dissolved in 6 mL acetonitrile/1-propanol, ratio 2:1. Thesolution was left overnight at ambient temperature to crystallize andthen the crystals were filtered off.

Method 18-H: Preparation of Crystalline Compound B,hemi-1,5-naphtalenedisulfonic acid salt

The sample from Method 18-C was dissolved in about 2 mL methanol.Ethanol (about 3 mL) was added as anti-solvent at ambient temperatureand seeds were added. No crystallization occurred, so solvents wereevaporated (about half of the amount) and a new portion of ethanol(about 2 mL) and seeds were added. Crystalline particles were formedwhen stirred at ambient temperature during night.

Method 18-I: Preparation of Crystalline Compound B,hemi-1,5-naphtalenedisulfonic acid salt

Amorphous Compound B (104.1 mg) was dissolved in 2-propanol and 1equivalent of 1,5-naphthalene-disulfonic acid tetrahydrate, dissolved in2-propanol, was added In total, the 2-propanol amount was about 2.5 mL.The solution was stirred at 44° C. for about 80 minutes and aprecipitate was formed. The particles were crystalline according topolarised light microscopy. The sample was filtered.

Method 18-J: Preparation of Crystalline Compound B,hemi-1,5-naphtalenedisulfonic acid salt

Compound B, hemi-1,5-naphtalenedisulfonic acid salt (56.4 mg) wasdissolved in 1.5 mL methanol. Methyl ethyl ketone (3 mL) was added.Seeds were added to the solution and crystallization started. Thecrystals were filtered off, washed with methyl ethyl ketone and airdried.

Method 18-K: Preparation of crystalline Compound B,hemi-1,5-naphtalenedisulfonic acid salt

Amorphous Compound B (161,0 mg) was dissolved in 3.5 mL 1-Butanol andthe solution was heated to 40° C. In another beaker 57.4 mg ofnaphthalene-disulfonic acid tetrahydrate was dissolved in 3 mL1-Butanol. A couple of drops of the acid solution were added to thesolution of compound B. Then seeds were added to the solution and after2 hours the rest of the acid solution was added (at 40° C.) slowly. Thenthe temperature was slowly decreased to room temperature and theexperiment was left under stirring overnight. The slurry was filtered,washed with 1-Butanol and dried under vacuum at 44° C. for 2 hours. Theyield was 83%.

Characterisation

Crystals of Compound B, hemi-1,5-naphtalenedisulfonic acid salt,obtained by way of Method 18-D above, was charaterised by NMR asfollows:

21.3 mg of the salt was dissolved in deuterated methanol, 0.7 ml wasinvestigated with NMR spectroscopy. A combination of 1D (¹H, ¹³C andselective NOE) and 2D (gCOSY, GHSQC and gHMBC) NMR experiments was used.

All data are in good agreement with the proposed structure, shown below.All carbons and the protons attached to carbons are assigned. Protonsattached to heteroatoms are exchanged for deuterium from the solvent andare not detected. Most resonances in the 1D ¹H and ¹³C NMR spectra arepresent as sets of two peaks. The reason for this is a slow rotationaround the C9-N10 bond, which results in two atropisomers thatsimultaneously exist in the solution. The 1D NOE experiment is anevidence for this. When a resonance of one atropisomer is irradiated,the saturation is transferred to the corresponding peak of the otheratropisomer. The resonances corresponding to the1,5-naphtalenedisulfonate counter ion do not show atropisomerism.

There are four fluorine atoms in the molecule. They give rise to splitresonances for some protons and carbons. Both the proton and the carbonresonance corresponding to position 1 are split due to the spincouplingwith the two fluorine nuclei in that position. The coupling constantsare ²J_(HF)=73 Hz and ¹J_(CF)=263 Hz. Further, the proton resonancecorresponding to H19 is a distorted doublet with ³J_(HF)=6.9 Hz due tothe spincoupling with the fluorine nuclei in position 18. Carbonresonances corresponding to C17, C18, C19 and C20 also exhibit couplingswith these fluorine nuclei. The C17 and C20 resonances are triplets with²J_(CF)=19 Hz and ³J_(CF)=11 Hz, respectively. The C18 resonance is adoublet of doublets with coupling constants ¹J_(CF)=251 Hz and ³J_(CF)=8Hz. The C19 resonance is a multiplet.

Comparing the magnitudes of integrals for resonances corresponding tothe 1,5-naphtalenedisulfonate counter ion and the mother compound givesthe stoichiometric relation of a single 1,5-naphtalenedisulfonatecounter ion crystallized with two molecules of the mother compound.

¹H and ¹³C NMR chemical shift assignment and proton-proton correlationsare shown in Table 11. TABLE 11 ¹³C Through-bond Atom shift/ ¹Hshift/ppm^(b) and correlation No. Typ ppm^(a) multiplicity^(c) J_(HH)/Hzto ¹H^(d)  1 CHF₂ 117.5^(e) 6.91 (t) 73 (²J_(HF)) nd  1′ 117.5^(e) 6.87(t) 73 (²J_(HF)) nd  2 C 153.5 na na na  2′ 153.3 na na na  3 CH 120.07.14 (t)^(n) nd 5, 7  3′ 119.6 7.11 (t)^(n) nd 5′, 7′  4 C 136.1 na nana  4′ 135.8 na na na  5 CH 125.0 7.31 (t)^(n) nd 3, 7  5′ 124.9 7.28(t)^(n) nd 3′, 7′  6 C 144.4 na na na  6′ 145.3 na na na  7 CH 117.27.16 (t)^(n) nd 3, 5  7′ 117.1 7.12 (t)^(n) nd 3′, 5′  8 CH 72.9 5.15(s) na nd  8′ 73.6 5.07 (s) na nd  9 CO 173.0 na na na  9′ 173.5 na nana 11 CH₂ 51.5 a: 4.29 (m) b: 4.13 nd 12, 13 11′ 48.6 (m) nd 12′, 13′ a:4.01 (m) b: 3.93 (m) 12 CH₂ 21.7 a: 2.46 (m) b: 2.17 nd 11, 13 12′ 22.8(m) nd 11′, 13′ a: 2.61 (m) b: 2.03 (m) 13 CH 62.8 4.70 (dd) 6.0 and 1213′ 65.8 5.14 (dd) 9.4 12′ 5.6 and 9.1 14 CO 172.4 na na na 14′ 173.2 nana na 16 CH₂ 32.3 4.51 (m) nd nd 16′ 32.5 4.51 (m) nd nd 17 C 121.0^(f)na na na 18 CF 162.8^(g) na na na 19 CH 112.7^(i) 7.35 (d) 6.9 (³J_(HF))nd 20 C 127.9^(k) na na na 21 C 160.0 na na na 21′ 159.9 na na na 24 CH₃64.8 3.93 (s) na nd 24′ 64.8 3.92 (s) na nd 25 C 142.4 na na na 26 CH126.8 8.16 (d) 7.2 27, 28 27 CH 125.9 7.54 (dd) 8.6 and 26, 28 7.2 28 CH131.0 8.97 (d) 8.6 26, 27 29 C 131.1 na na na^(a)Relative to the solvent resonance at 49.0 ppm.^(b)Relative to the solvent resonance at 3.30 ppm.^(c)s = singlet, d = doublet, dd = doublet of doublets, t = triplet, m =multiplet.^(d)Obtained in the gCOSY experiment.^(e)The resonance is a triplet due to coupling with the two fluorinenuclei F1.¹J_(CF) = 263 Hz.^(f)The resonance is a triplet due to coupling to the two fluorinenuclei F18.²J_(CF) = 19 Hz.^(g)The resonance is a doublet of doublets due to coupling to the twofluorine nuclei F18.¹J_(CF) = 251 Hz and³J_(CF) = 8 Hz.^(i)The resonance is a multiplet due to coupling to the two fluorinenuclei F18.^(k)The resonance is a triplet due to coupling to the two fluorinenuclei F18.³J_(CF) = 11 Hz.^(n)The ⁴J_(HH) coupling with the meta-protons is not fully resolved.na = not applicable,nd = not determined

Crystals of Compound B, hemi-1,5-naphtalenedisulfonic acid salt(obtained by way of Method 18-I above, were analyzed by XRPD and theresults are tabulated below (Table 12) and are shown in FIG. 5. TABLE 12Intensity d value (Å) (%) Intensity 18.3 99 vs 12.5 22 s 9.9 22 s 9.1 67vs 8.0 18 m 7.5 17 m 6.8 37 s 6.7 59 s 6.1 39 s 6.0 21 s 5.6 66 vs 5.598 vs 4.94 48 s 4.56 59 s 4.39 35 S 4.27 33 s 4.13 81 vs 4.02 87 vs 3.8688 vs 3.69 69 vs 3.63 100 vs 3.57 49 s 3.48 53 s 3.23 35 s 3.19 43 s3.16 38 s

DSC showed an endotherm with an extrapolated melting onset temperatureof ca 183° C. and TGA showed a 0.3% weight loss between 25-110° C.

Abbreviations

-   Ac=acetyl-   APCI=atmospheric pressure chemical ionisation (in relation to MS)-   API=atmospheric pressure ionisation (in relation to MS)-   aq.=aqueous-   Aze(& (S)-Aze)=(S)-azetidine-2-carboxylate (unless otherwise    specified)-   Boc=tert-butyloxycarbonyl-   br=broad (in relation to NMR)-   CI=chemical ionisation (in relation to MS)-   d=day(s)-   d=doublet (in relation to NMR)-   DCC=dicyclohexyl carbodiimide-   dd=doublet of doublets (in relation to NMR)-   DIBAL-H=di-isobutylaluminium hydride-   DIPEA=diisopropylethylamine-   DMAP=4-(N,N-dimethyl amino) pyridine-   DMF=N,N-dimethylformamide-   DMSO=dimethylsulfoxide-   DSC=differential scanning colorimetry-   DVT=deep vein thrombosis-   EDC=1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride-   eq.=equivalents-   ES=electrospray-   ESI=electrospray interface-   Et=ethyl-   ether=diethyl ether-   EtOAc=ethyl acetate-   EtOH=ethanol-   Et₂O=diethyl ether-   HATU=O-(azabenzotriazol-1-yl)-N,N′,N′-tetramethyluronium    hexafluorophosphate-   HBTU=[N,N,N′,N′-tetramethyl-O-(benzotriazol-1-yl)uronium    hexafluorophosphate]-   HCl=hydrochloric acid, hydrogen chloride gas or hydrochloride salt    (depending on context)-   Hex=hexanes-   HOAc=acetic acid-   HPLC=high performance liquid chromatography-   LC=liquid chromatography-   m=multiplet (in relation to NMR)-   Me=methyl-   MeOH=methanol-   min.=minute(s)-   MS=mass spectroscopy-   MTBE=methyl tert-butyl ether-   NMR=nuclear magnetic resonance-   OAc=acetate-   Pab=para-amidinobenzylamino-   H-Pab=para-amidinobenzylamine-   Pd/C=palladium on carbon-   Ph=phenyl-   PyBOP=(benzotriazol-1-yloxy)tripyrrolidinophosphonium    hexafluorophosphate-   q=quartet (in relation to NMR)-   QF=tetrabutylammonium fluoride-   rt/RT=room temperature-   s=singlet (in relation to NMR)-   solutol=PEG 660 12-hydroxy stearate (a non-ionic surfactant)-   t=triplet (in relation to NMR)-   TBTU=[N,N,N′,N′-tetramethyl-O-(benzotriazol-1-yl)uronium    tetrafluoroborate]-   TEA=triethylamine-   Teoc=2-(trimethylsilyl)ethoxycarbonyl-   TEMPO=2,2,6,6-tetramethyl-1-piperidinyloxy free radical-   TFA=trifluoroacetic acid-   TGA=thermogravimetric analysis-   THF=tetrahydrofuran-   TLC=thin layer chromatography-   UV=ultraviolet

Prefixes n-, s-, i-, t- and tert- have their usual meanings: normal,secondary, iso, and tertiary.

The invention is illustrated by way of the following Examples.

EXAMPLE 1

Compound A 30 μmol PEG 400/ethanol/water 50/5/45 (w/w) % to 1 mL

A formulation was prepared by dissolving Compound A in PEG400/ethanol/water 50/5/45 (w/w) % followed by gently stirring. Thiscomposition was given to dogs orally by gavage once daily for 5 days.The dose 150 μmol/kg gave maximum plasma concentrations in the range118-254 μM (118-254 μmol/L) after the first dose and 186-286 μM (186-286μmol/L) after the fifth dose.

EXAMPLE 2

Compound A 40 μmol PEG 400/ethanol/water 50/5/45 (w/w) % to 1 mL

A formulation was prepared by dissolving Compound A in PEG400/ethanol/water 50/5/45 (w/w) % followed by gently stirring. Thiscomposition was given to rats orally by gavage once daily for 5 days.The dose 400 μmol/kg gave maximum plasma concentrations in the range3.17-6.91 μM (3.17-6.91 μmol/L) after the first dose and 3.01-10.5 μM(3.01-10.5 μmol/L) after the fifth dose.

EXAMPLE 3

Compound A 80 μmol PEG 400/ethanol/water 50/5/45 (w/w) % to 1 mL

A formulation was prepared by dissolving Compound A in PEG400/ethanol/water 50/5/45 (w/w) % followed by gently stirring. Thiscomposition was given to rats orally by gavage once daily for 5 days.The dose 800 μmol/kg gave maximum plasma concentrations in the range7.00-23.9 μM (7.00-23.9 μmol/L) after the first dose and 10.3-32.8 μM(10.3-32.8 μmol/L) after the fifth dose.

EXAMPLE 4

Compound A 250 μmol PEG 400/ethanol/water 50/5/45 (w/w) % to 1 mL

A formulation was prepared by dissolving Compound A in PEG400/ethanol/water 50/5/45 (w/w) % followed by gently stirring. Thesolubility of Compound A is at least 1000 times higher in this vehiclecompared to water alone.

EXAMPLE 5

Compound A 21 μmol PEG 400/ethanol/water 20/10/70 (w/w) % to 1 mL

A formulation was prepared by dissolving Compound A in PEG400/ethanol/water 20/10/70 (w/w) % followed by gently stirring. Thesolubility of Compound A is at least 100 times higher in this vehiclecompared to water alone.

EXAMPLE 6

Compound A 51 μmol PEG 400/ethanol/water 20/10/70 (w/w) % to 1 mLThe water contained 50 μmol/mL Tartaric Acid

A formulation was prepared by dissolving Compound A in acidified PEG400/ethanol/water 20/10/70 (w/w) % that was followed by gently stirring.The pH of this solution was 3.6. The solubility of Compound A is atleast 250 times higher in this vehicle compared to water alone.

EXAMPLE 7

Compound A 44 μmol PEG 400/ethanol/water 30/5/65 (w/w) % to 1 mL

A formulation was prepared by dissolving Compound A in PEG400/ethanol/water 30/5/65 (w/w) % followed by gently stirring. Thesolubility of Compound A is at least 200 times higher in this vehiclecompared to water alone.

EXAMPLE 8

Compound A 88 μmol PEG 400/ethanol/water 30/5/65 (w/w) % to 1 mL Thewater contained 50 μmol/mL Tartaric Acid q.s. HCl to pH 3.6

A formulation was prepared by dissolving Compound A in acidified PEG400/ethanol/water 30/5/65 (w/w) % followed by gently stirring. The pH ofthis solution was set to 3.6 by addition of HCl. The solubility ofCompound A is at least 400 times higher in this vehicle compared towater alone.

EXAMPLE 9

Compound A 120 μmol PEG 400/ethanol/water 40/5/55 (w/w) % to 1 mL

A formulation was prepared by dissolving Compound A in PEG400/ethanol/water 40/5/55 (w/w) % followed by gently stirring. Thesolubility of Compound A is at least 600 times higher in this vehiclecompared to water alone.

EXAMPLE 10

Compound A 198 μmol PEG 400/ethanol/water 40/5/55 (w/w) % to 1 mL Thewater contained 50 μmol/mL Tartaric Acid q.s. HCl to pH 3.8

A formulation was prepared by dissolving Compound A in acidified PEG400/ethanol/water 40/5/55 (w/w) % followed by gently stirring. The pH ofthis solution was set to 3.8 by addition of HCl. The solubility ofCompound A is at least 1000 times higher in this vehicle compared towater alone. Formulations of Compound A in this vehicle are stable forat least 3 months at <−15° C.

EXAMPLE 11

Compound A 136 μmol Hydroxypropyl-β-cyclodextrin/water 40/60 (w/w) % to1 mL HCl to pH 3.7 q.s.

A formulation was prepared by dissolving Compound A inHydroxypropyl-β-cyclodextrin/water 40/60 (w/w) % followed by gentlystirring. The pH of this solution was set to 4.7 by addition of HCl. Thesolubility of Compound A is at least 700 times higher in this vehiclecompared to water alone.

EXAMPLE 12

Compound A 76 μmol Hydroxypropyl-β-cyclodextrin/water 28/72 (w/w) % to 1mL

A formulation was prepared by dissolving Compound A inHydroxypropyl-β-cyclodextrin/water 28/72 (w/w) % followed by gentlystirring. The solubility of Compound A is at least 400 times higher inthis vehicle compared to water alone.

EXAMPLE 13

Compound A 40 μmol PEG 400/ethanol/solutol ™/water 50/5/5/40 (w/w) % to1 mL

A formulation was prepared by dissolving Compound A in PEG400/ethanol/solutol™/water 50/5/5/40 (w/w) % followed by gentlystirring. The solubility of Compound A is at least 80 times higher inthis vehicle compared to water alone.

EXAMPLE 14

Compound A 40 μmol PEG 400/water 40/60 (w/w) % to 1 mL

A formulation was prepared by dissolving Compound A in PEG 400 followedby gently stirring for at least 1 hour, thereafter water was added tothe final volume. The solubility of Compound A is at least 200 timeshigher in this vehicle compared to water alone.

EXAMPLE 15

Compound A 52 μmol PEG 400/water 35/65 (w/w) % to 1 mL The watercontained 50 μmol/mL Tartaric Acid

A formulation was prepared by dissolving Compound A in PEG 400 followedby gently stirring for at least 1 hour, thereafter water was added tothe final volume. The solubility of Compound A is at least 250 timeshigher in this vehicle compared to water alone.

EXAMPLE 16

Compound A 58 μmol PEG 400/water 50/50 (w/w) % to 1 mL

A formulation was prepared by dissolving Compound A in PEG 400 followedby gently stirring for at least 1 hour, thereafter water was added tothe final volume. The solubility of Compound A is at least 300 timeshigher in this vehicle compared to water alone.

EXAMPLE 17

Compound A 88 μmol PEG 400/water 67/33 (w/w) % to 1 mL

A formulation was prepared by dissolving Compound A in PEG 400 followedby gently stirring for at least 1 hour, thereafter water was added tothe final volume. The solubility of Compound A is at least 400 timeshigher in this vehicle compared to water alone.

EXAMPLE 18

Compound A 92 μmol PEG 400/ethanol/water 45/1/54 (w/w) % to 1 mL

A formulation was prepared by dissolving Compound A in PEG400/ethanol/water 45/1/54 (w/w) % followed by gently stirring. Thesolubility of Compound A is at least 450 times higher in this vehiclecompared to water alone.

EXAMPLE 19

Compound A 159 μmol PEG 400/ethanol/water 45/1/54 (w/w) % to 1 mL Thewater contained 50 μmol/mL Tartaric Acid q.s. HCl to pH 4.2

A formulation was prepared by dissolving Compound A in acidified PEG400/ethanol/water 45/1/54 (w/w) % followed by gently stirring. The pH ofthis solution was set to 4.2 with HCl. The solubility of Compound A isat least 800 times higher in this vehicle compared to water alone.

EXAMPLE 20

Compound A 101 μmol PEG 400/ethanol/water 45/2/53 (w/w) % to 1 mL

A formulation was prepared by dissolving Compound A in PEG400/ethanol/water 45/2/53 (w/w) % followed by gently stirring. Thesolubility of Compound A is at least 500 times higher in this vehiclecompared to water alone.

EXAMPLE 21

Compound A 167 μmol PEG 400/ethanol/water 45/2/53 (w/w) % to 1 mL Thewater contained 50 μmol/mL Tartaric Acid q.s. HCl to pH 4.3

A formulation was prepared by dissolving Compound A in acidified PEG400/ethanol/water 45/2/53 (w/w) % followed by gently stirring. The pH ofthis solution was set to 4.3 by addition of HCl. The solubility ofCompound A is at least 800 times higher in this vehicle compared towater alone.

EXAMPLE 22

Compound A 46 μmol DMA/water 50/50 (w/w) % to 1 mL

A formulation was prepared by dissolving Compound A in the vehiclefollowed by gently stirring for at least 1 hour. The solubility ofCompound A is at least 230 times higher in this vehicle compared towater alone.

EXAMPLE 23

Compound A 29 μmol DMA/water 25/75 (w/w) % to 1 mL

A formulation was prepared by dissolving Compound A in the vehiclefollowed by gently stirring for at least 1 hour. The solubility ofCompound A is at least 150 times higher in this vehicle compared towater alone.

EXAMPLE 24

Compound A 5 μmol HCl 10 μmol Water to 1 mL HCl/NaOH to pH 3.6 q.s.

A formulation was prepared by dissolving Compound A in a lower volume ofthe double equimolar amount of HCl followed by gently stirring anddilution to 1 mL. The pH of the final solution was adjusted to 3.6. Thesolubility of Compound A is at least 20 times higher in this vehiclecompared to water alone.

EXAMPLE 25

Compound A 10 μmol Water to 1 mL HCl to pH 1.0 q.s. NaOH to pH 3.0 q.s.

A formulation was prepared by dissolving Compound A water and HCl wasadded to give pH 1 thereafter the solution was gently stirred. The pH ofthe final solution was adjusted to 3.0 with NaOH. The solubility ofCompound A is at least 40 times higher in this vehicle compared to wateralone. This formulation was given p.o to rats in a kinetic comparativestudy.

EXAMPLE 26

Compound A 100 μmol Miglyol 0.25 g/g Compound A DMA to 1 mL

A formulation was prepared by dissolving Compound A in 1 mL DMA/miglyolfollowed by gently stirring. The solubility of Compound A is at least4000 times higher in this vehicle compared to water alone.

EXAMPLE 27

Compound A 100 μmol Miglyol 0.25 g/g Compound A Ethanol to 1 mL

A formulation was prepared by dissolving Compound A in 1 mLEthanol/Miglyol followed by gently stirring. The solubility of CompoundA is at least 4000 times higher in this vehicle compared to water alone.

EXAMPLE 28

Compound A 130 μmol Ethanol to 1 mL

A formulation was prepared by dissolving Compound A in 1 mL ethanolfollowed by gently stirring. The substance is stable in this formulationmore than 1 week.

EXAMPLE 29

In order to prepare nanoparticles a stock solution of Compound A ofabout 100 mM in ethanol was used. Included was also 25% (w/w) Miglyol,calculated on the amount of the substance. The solutions were diluted1/10 with the stabilizer solution, consisting of 0.2% (w/w) PVP and 0.25mM SDS in water. The mixing, which is considered as a critical parameterduring the nanoparticle preparation, was rapid and instant. The drugsolution was rapidly injected into the stabilizer solution duringultrasonication. After the 1/10 dilution in the aqeous solution,nanoparticles of about 150 nm were achieved. After 6 hours at roomtemperature, the particle sizes were unchanged.

EXAMPLE 30

Compound A 4 μmol saline/ethanol/solutol 90/5/5 (w/w) % to 1 mL

A formulation was prepared by dissolving Compound A insaline/ethanol/solutol 90/5/5 (w/w) % followed by gently stirring. Thesolution was given orally to rats and the plasma concentration ofCompound D was 0.56 μmol/L after 1 hour. The solution was givensubcutaneously to rats and the plasma concentrations of Compound D and Awere 0.24 μmol/L and 0.6 μmol/L, respectively, after 1 hour.

EXAMPLE 31

Compound B 4 μmol saline/ethanol/solutol 90/5/5 (w/w) % to 1 mL

A formulation was prepared by dissolving Compound B insaline/ethanol/solutol 90/5/5 (w/w) % followed by gently stirring. Thesolution was given orally to rats and the plasma concentrations ofCompound B and Compound E were respectively 0.07 μmol/L and 0.65 μmol/L,after 1 hour. The solution was given subcutaneously to rats and theplasma concentrations of Compound B and E were 0.4 μmol/L and 0.3μmol/L, respectively, after 1 hour.

EXAMPLE 32

Compound C 4 μmol saline/ethanol/solutol 90/5/5 (w/w) % to 1 mL

A formulation was prepared by dissolving Compound C insaline/ethanol/solutol 90/5/5 (w/w) % followed by gently stirring. Thesolution was given orally to rats and the plasma concentrations ofCompounds C and F were respectively 0.2 μmol/L and 0.5 μmol/L after 1hour. The solution was given subcutaneously to rats and the plasmaconcentrations of Compounds C and F were 0.35 μmol/L and 0.5 μmol/L,respectively, after 1 hour.

EXAMPLE 33

Compound D (trifluoroacetate salt) 5 μmol Saline 9 mg/ml to 1 mL

A formulation was prepared by dissolving the salt of Compound D in 1 mLsaline followed by gently stirring.

EXAMPLE 34

Compound D (trifluoroacetate salt) 75 μmol EtOH 0.05 mL Saline(9 mg/ml)to 1 mL

A formulation was prepared by dissolving the salt of Compound D in 1 mLsaline/ethanol solution followed by gently stirring.

EXAMPLE 35

Compound D (trifluoroacetate salt) 4 μmol EtOH 0.02 mL saline to 1 mL

A formulation was prepared by dissolving the salt of Compound D in 1 mLsaline/etanol solution followed by gently stirring. The solution wasgiven subcutaneously to rats and the plasma concentration of Compound Dwas 0.55 μmol/L after 1 hour.

EXAMPLE 36

Compound E (acetate salt) 4 μmol EtOH 0.02 mL saline to 1 mL

A formulation was prepared by dissolving the salt of Compound E in 1 mLsaline/ethanol solution followed by gently stirring. The solution wasgiven subcutaneously to rats and the plasma concentration of Compound Ewas 0.75 μmol/L after 1 hour.

EXAMPLE 37

Compound F (trifluoroacetate salt) 4 μmol EtOH 0.02 mL saline to 1 mL

A formulation was prepared by dissolving the salt of Compound F in 1 mLsaline/ethanol solution followed by gently stirring. The solution wasgiven subcutaneously to rats and the plasma concentration Compound F was0.92 μmol/L after 1 hour.

EXAMPLE 38

Compound E (acetate salt) 22 mg Saline 9 mg/ml to 1 mL

A formulation was prepared by dissolving the salt of Compound E in 1 mLsaline followed by gently stirring.

EXAMPLE 39

Compound F (trifluoroacetate salt) 22 mg Saline 9 mg/ml to 1 mL

A formulation was prepared by dissolving the salt of Compound F in 1 mLsaline followed by gently stirring.

EXAMPLE 40

Compound A (as esylate salt) 14 mg water to 1 mL

A solution was prepared by dissolving excess of Compound A as esylatesalt in 3 mL water followed by gently stirring over night. A finalconcentration of the solution after filtration was monitored to 14 mg/mlat a pH of 2.7.

EXAMPLE 41

Compound A (as esylate salt) 33 mg Sodium phosphate buffer pH = 3.1 I =0.1 to 1 mL

A solution was prepared by dissolving 112 mg of Compound A as esylatesalt in 3 mL sodium phosphate buffer followed by gently stirring overnight. A final concentration of the solution after filtration wasmonitored to 33 mg/ml at a pH of 2.7.

EXAMPLE 42

Compound A (as esylate salt) 1.6 mg Sodium phosphate buffer pH = 6.9 I =0.1 to 1 mL

A solution was prepared by dissolving 20 mg of Compound A as esylatesalt in 3 mL sodium phosphate buffer followed by gently stirring overnight. A final concentration of the solution after filtration wasmonitored to 1.6 mg/ml at a pH of 6.5.

EXAMPLE 43

The following freeze dried formulations can be made in accordance withtechniques described in one or more of Examples 1-29 above: a. CompoundA 10 μmol Mannitol 10 mg Water to 1 mL HCl to pH 1.0 q.s. NaOH to pH 3.0q.s. b. Compound D 10 μmol Mannitol 10 mg Water to 1 mL HCl to pH 1.0q.s. NaOH to pH 3.0 q.s. c. Compound E 10 μmol Mannitol 10 mg Water to 1mL HCl to pH 1.0 q.s. NaOH to pH 3.0 q.s. d. Compound F 10 μmol Mannitol10 mg Water to 1 mL HCl to pH 1.0 q.s. NaOH to pH 3.0 q.s. e. Compound B10 μmol Mannitol 10 mg Water to 1 mL HCl to pH 1.0 q.s. NaOH to pH 3.0q.s. f. Compound C 10 μmol Mannitol 10 mg Water to 1 mL HCl to pH 1.0q.s. NaOH to pH 3.0 q.s. g. Compound A (as esylate salt) 14 mg Mannitol10 mg Water to 1 mL HCl to pH 1.0 q.s. NaOH to pH 3.0 q.s. h. Compound A(as besylate salt) 14 mg Mannitol 10 mg Water to 1 mL HCl to pH 1.0 q.s.NaOH to pH 3.0 q.s.

The solutions are optionally sterile filtered, for example through a0.22 μm membrane filter. Solutions (sterile or otherwise) are filledinto appropriate vessels (e.g. vials) and the formulations arefreeze-died using standard equipment. Vials may be sealed infreeze-dryer equipment under a nitrogen atmosphere.

EXAMPLE 44

Weight Amount Compound A 48 mg 17% Polyvinyl pyrrolidone K90  8 mg 3%Mannitol 21 mg 7% Microcrystalline cellulose 187 mg  65% Sodium starchglycolate 21 mg 7% Sodium stearyl fumarate  3 mg 1%

The excipients and drug were mixed and granulated with polyvinylpyrrolidone K90 dissolved in water. The granules were then dried in adrying oven. The granulate was lubricated with sodiumstearylfumarate andcompressed into tablets using an excenterpress.

Three individual tablets were tested for drug release in 900 ml mediausing a USP dissolution apparatus 2 (paddle+basket¹) at 50 rpm and 37°C. The dissolution media used were 0.1 M hydrochloric acid (pH 1) and0.1 M sodium phosphate buffer (pH 6.8). In-line quantitation wasperformed using the C Technologies fibre optic system with 220 nm as theanalytical wavelength when 0.1 M HCl was used as the dissolution mediaand with 260 nm as the analytical wavelength when phosphate buffer pH6.8 was used as the dissolution media. 350 nm was used as the referencewavelength with both media. For the first two hours of the analysis therelease value was measured every 15 minutes, and then every hour for theremainder of the analysis. The results are presented in the table below.[¹ A custom made quadrangular basket of mesh wire, soldered in one ofits upper, narrow sides to the end of a steel rod. The rod is broughtthrough the cover of the dissolution vessel and fixed by means of twoTeflon nuts, 3.2 cm from the centre of the vessel. The lower edge of thebottom of the basket is adjusted to be 1 cm above the paddle. The basketis directed along the flow stream with the tablet under test standing onits edge].% released in % released in Time (min) buffer pH 1.1 buffer pH 6.8 0 0 015 100 44 30 100 49 45 100 51 60 100 53 120 100 57 180 100 61 240 100 63360 100 67 480 100 70 600 100 75 720 100 77 840 100 79 960 100 82 1080100 83 1200 100 86

EXAMPLE 45

Weight Amount Esylate salt of Compound A 58 mg 20% Polyvinyl pyrrolidoneK90  8 mg 3% Mannitol 21 mg 7% Microcrystalline cellulose 177 mg  62%Sodium starch glycolate 21 mg 7% Sodium stearyl fumarate  3 mg 1%

The excipients and drug were mixed and granulated with polyvinylpyrrolidone K90 dissolved in water. The granules were then dried in adrying oven. The granulate was lubricated with sodium stearyl fumarateand compressed into tablets using an excenterpress.

EXAMPLE 46

Weight Amount Compound B 48 mg 17% Polyvinyl pyrrolidone K90  8 mg 3%Mannitol 21 mg 7% Microcrystalline cellulose 187 mg  65% Sodium starchglycolate 21 mg 7% Sodium stearyl fumarate  3 mg 1%

The excipients and drug were mixed and granulated with polyvinylpyrrolidone K90 dissolved in water. The granules were then dried in adrying oven. The granulate was lubricated with sodium stearyl fumarateand compressed into tablets using an excenterpress

EXAMPLE 47

Weight Amount Compound C 48 mg 17% Polyvinyl pyrrolidone K90  8 mg 3%Mannitol 21 mg 7% Microcrystalline cellulose 187 mg  65% Sodium starchglycolate 21 mg 7% Sodium stearyl fumarate  3 mg 1%

The excipients and drug were mixed and granulated with polyvinylpyrrolicdone K90 dissolved in water. The granules were then dried in adrying oven. The granulate was lubricated with sodium stearyl fumarateand compressed into tablets using an excenterpress

EXAMPLE 48

Compound A 16 μmol PEG 414 to 1 mL

A formulation was prepared by dissolving Compound A in acidified PEG414followed by gently stirring.

EXAMPLE 49

Compound A 16 μmol PEG 300 to 1 mL

A formulation was prepared by dissolving Compound A in acidified PEG300followed by gently stirring.

EXAMPLE 50

Compound A 16 μmol PEG 200 to 1 mL

A formulation was prepared by dissolving Compound A in acidified PEG200followed by gently stirring.

EXAMPLE 51

Compound G 4 μmol saline/ethanol/solutol 90/5/5 (w/w) % to 1 mL

A formulation was prepared by dissolving Compound G insaline/ethanol/solutol 90/5/5 (w/w) % followed by gently stirring.

EXAMPLE 52

Compound J 4 μmol saline/ethanol/solutol 90/5/5 (w/w) % to 1 mL

A formulation was prepared by dissolving Compound J insaline/ethanol/solutol 90/5/5 (w/w) % followed by gently stirring.

EXAMPLE 53

Compound H 4 μmol saline/ethanol/solutol 90/5/5 (w/w) % to 1 mL

A formulation was prepared by dissolving Compound H insaline/ethanol/solutol 90/5/5 (w/w) % followed by gently stirring.

EXAMPLE 54

Weight Amount Compound A esylate salt 500 mg 66% Polyvinyl pyrrolidoneK30 100 mg 13% Microcrystalline cellulose 100 mg 13% Crosslinked sodiumCMC  50 mg 7% Magnesium stearate  5 mg 1%

Formulation can be prepared in accordance with Example 47 above.

EXAMPLE 55

Weight Amount Compound A n-propane 100 mg 23% sulphonic acid saltPolyvinyl pyrrolidone K30  60 mg 14% Lactose monohydrate 100 mg 23%Microcrystalline cellulose 150 mg 34% Polyvinyl pyrrolidone  20 mg 5%crosslinked Sodium stearyl fumarate  10 mg 2%

Formulation can be prepared in accordance with Example 47 above.

EXAMPLE 56

Weight Amount Compound A besylate salt 20 mg 8% Hydroxypropyl cellulose15 mg 6% Microcrystalline cellulose 200 mg  79% Crosslinked sodium CMC15 mg 6% Sodium stearyl fumarate  3 mg 1%

Formulation can be prepared in accordance with Example 47 above.

EXAMPLE 57

Compound A 24 μmol PEG 400/ethanol/water 25/10/65 (w/w) % to 1 mL

A formulation was prepared by dissolving Compound A in PEG400/ethanol/water 25/10/65 (w/w) % followed by gently stirring. Thesolubility of Compound A is at least 100 times higher in this vehiclecompared to water alone. The formulation is stable in a freezer for atleast 2 months.

EXAMPLE 58

Compound A 800 μmol PEG 400/ethanol/water 50/10/40 (w/w) % to 1 mL

A formulation was prepared by dissolving Compound A in PEG400/ethanol/water 50/10/40 (w/w) % followed by gently stirring. Thesolubility of Compound A is at least 2000 times higher in this vehiclecompared to water alone.

EXAMPLE 59

Compound A 500 μmol Citric acid 200 μmol HCl to pH 3.6 q.s. PEG400/ethanol/9 mg/ml NaCl 40/10/50 (w/w) % to 1 mL

A formulation was prepared by dissolving Compound A in PEG400/ethanol/water 40/10/50 (w/w) % followed by gently stirring. Thesolubility of Compound A is at least 1500 times higher in this vehiclecompared to water alone.

EXAMPLE 60

Compound A 24 μmol citric acid 5 μmol HCl to pH 3.2 q.s. ethanol/water12/88 (w/w) % to 1 mL

A formulation was prepared by dissolving Compound A in ethanol followedby gently stirring, thereafter citric acid and water was added to finalvolume and the pH was set to 3.2. The solubility of Compound A is atleast 100 times higher in this vehicle compared to water alone. Theformulation is stable in a freezer for at least 1 month.

EXAMPLE 61

Compound A 2 μmol citric acid 5 μmol HCl to pH 3.6 q.s. 9 mg/ml NaCl to1 mL

A formulation was prepared by dissolving Compound A and citric acid inphysicological saline followed by gently stirring. The pH was set to3.6. The formulation is stable in a freezer for at least 3 months.

EXAMPLE 62

Compound A (as besylate salt) 140 μmol citric acid 5 μmol HCl to pH 3.6q.s. PEG 400/ethanol/water 40/5/55 (w/w) % to 1 mL

A formulation was prepared by dissolving Compound A in PEG400/ethanol/water 40/5/55 (w/w) % containing citric acid followed bygently stirring and setting the pH to 3.6. The formulation is stable ina freezer for at least 1 month.

EXAMPLE 63

Compound A (as besylate salt) 65 μmol citric acid 5 μmol HCl to pH 3.3q.s. PEG 400/ethanol/water 20/5/75 (w/w) % to 1 mL

A formulation was prepared by dissolving Compound A in PEG400/ethanol/water 20/5/75 (w/w) % containing citric acid followed bygently stirring and the pH was set to 3.2.

EXAMPLE 64

Compound D (as acetate salt) 25 μmol PEG 400/ethanol/water 40/5/55 (w/w)% to 1 mL Tartaric Acid: Component A (acetate salt of D) q.s. equimolaramount plus 5 mM excess HCl to pH 3.6

A formulation was prepared by dissolving Compound D in acidified PEG400/ethanol/water 40/5/55 (w/w) % followed by gently stirring. The pH ofthis solution was set to 3.6 by addition of HCl. Formulations of D inthis vehicle are stable for at least 2 months at <−15° C.

EXAMPLE 65

Compound A 50 mg HPMC (15000 Cps) 5 mg Solutol HS15 20 mg Water to 1 mL

The HPMC was suspended in hot water and melted Solutol was added duringvigourous stirring. This solution was chilled and Compound A was addedunder vigourous stirring to form a well dispersed suspension.

EXAMPLE 66

Compound A (as besylate salt) 50 mg HPMC (15000 Cps) 5 mg Solutol HS1520 mg Water to 1 mL

The HPMC was suspended in hot water and melted Solutol was added duringvigourous stirring. This solution was shilled and Compound A (besylate)was added under vigourous stirring to form a well dispersed suspension.

EXAMPLE 67

Compound D (as acetate salt) 2 μmol citric acid 5 μmol HCl to pH 3.6q.s. 9 mg/ml NaCl to 1 mL

A formulation was prepared by dissolving Compound A and citric acid inphysicological saline and stirring gently. The pH was set to 3.6. Theformulation is stable in a freezer for at least 3 months.

EXAMPLE 68

To prepare nanoparticles a stock solution of Compound B of about 100 mMin ethanol was used. Included was also 25% (w/w) Miglyol, calculated onthe amount of the substance. The solutions were diluted 1/10 with astabilizer solution consisting of 0.2% (w/w) PVP and 0.25 mM SDS inwater. The critical mixing stage was rapid and instant:—The drugsolution was rapidly injected into the stabilizer solution duringultrasonication. After 1/10 dilution in the aqeous solution,nanoparticles of about 110 nm were obtained. After 6 hours at roomtemperature, the particle sizes were unchanged.

Optionally DMA may be used instead of ethanol, Miglyol may be excludedand the dilution may be larger (1/20). Particles in the size range 100to 300 nm may be obtained by different combinations.

EXAMPLE 69

Compound B 200 μmol PEG 400/ethanol/water 50/5/45 (w/w) % to 1 mL

A formulation was prepared by dissolving Compound B in PEG400/ethanol/water 50/5/45 (w/w) % followed by gently stirring.Formulations of B (at 0.5 mg/mL) in this vehicle are stable for at least1 month at <−15° C.

EXAMPLE 70

Compound B 230 μmol PEG 400/ethanol/water 60/5/35 (w/w) % to 1 mL

A formulation was prepared by dissolving Compound B in PEG400/ethanol/60/5/35 (w/w) % followed by gently stirring.

EXAMPLE 71

Compound B 50 mg HPMC (15000 Cps) 5 mg Solutol HS15 20 mg Water to 1 mL

The HPMC was suspended in hot water and melted solutol was added duringvigourous stirring. This solution was chilled and Compound B was addedunder vigourous stirring to form a well dispersed suspension.

EXAMPLE 72

Compound E (as acetate salt) 39 μmol 9 mg/ml NaCl to 1 mL

A formulation was prepared by dissolving Compound E in 9 mg/ml NaCl bygently stirring. The pH obtained in this formulation is 8-9.

EXAMPLE 73

Compound C 400 μmol PEG 400/ethanol/water 50/5/45 (w/w) % to 1 mL

A formulation was prepared by dissolving Compound C in PEG400/ethanol/water 50/5/45 (w/w) % followed by gently stirring.Formulations of C (at 0.5 mg/mL) in this vehicle are stable for at least1 month at room temperature and below.

EXAMPLE 74

Compound C 16 μmol Hydroxypropyl-β-cyclodextrin/water 20/80 (w/w) % to 1mL

A formulation was prepared by dissolving Compound C inHydroxypropyl-β-cyclodextrin/water 20/80 (w/w) % followed by gentlystirring. Formulations of C in this vehicle are stable for at least 2weeks at <8° C.

EXAMPLE 75

Compound F (as trifluoroacetate salt) 38 μmol 9 mg/ml NaCl to 1 mL

A formulation was prepared by dissolving Compound F in 9 mg/ml NaCl bygently stirring. The pH obtained in this formulation is 3-4.Formulations of F in this vehicle are stable for at least 2 weeks at atroom temperature and below.

EXAMPLE 76

A tablet was prepared according to the general method of Example 44.Weight Amount Besylate salt of Compound A 66 mg 17% Polyvinylpyrrolidone K90  9 mg 2% Mannitol 29 mg 7% Microcrystalline cellulose256 mg  65% Sodium starch glycolate 29 mg 7% Sodium stearyl fumarate  4mg 1%

Release Data

Measured according to the general method of Example 44 but using 500 mlof media and 75 rpm. % released in Time (min) buffer pH 6.8 0 0 5 90 1094 15 96 20 96 30 98 45 98 60 100

EXAMPLE 77

A tablet is prepared according to the general method of Example 44.Weight Amount Besylate salt of Compound A 200 mg  40% Polyvinylpyrrolidone K30 10 mg 2% Lactose 200 mg  40% Microcrystalline cellulose70 mg 14% Polyvinylpolypyrrolidone CL 15 mg 3% Magnesium stearate  5 mg1%

Other formulations in which the quantity of the besylate salt ofCompound A is in the range 50-300 mg may be prepared; the ratio of othercomponents being similar to those in Example 77.

EXAMPLE 78

A tablet is prepared according to the general method of Example 44.Weight Amount Hemi-Naphthalene 1,5-disulphonic 48 mg 17% acid salt ofCompound B Polyvinyl pyrrolidone K90  8 mg 3% Mannitol 21 mg 7%Microcrystalline cellulose 187 mg  65% Sodium starch glycolate 21 mg 7%Sodium stearyl fumarate  3 mg 1%

Other formulations in which 100 mg or 200 mg of the hemi-naphthalene1,5-disulphonic acid salt of Compound B is used may also be prepared;the ratio of other components being similar to those in Example 78.

Particular aspects of the invention are provided as follows:—

-   1. An immediate release pharmaceutical formulation comprising, as    active ingredient, a compound of formula (I):    -   wherein    -   R¹ represents C₁₋₂ alkyl substituted by one or more fluoro        substituents;    -   R² represents hydrogen, hydroxy, methoxy or ethoxy; and    -   n represents 0, 1 or 2;    -   or a pharmaceutically acceptable salt thereof; and a        pharmaceutically acceptable diluent or carrier;    -   provided that the formulation does not solely contain:        -   a solution of one active ingredient and water;        -   a solution of one active ingredient and dimethylsulphoxide;            or,        -   a solution of one active ingredient in a mixture of            ethanol:PEG 660 12-hydroxy stearate:water 5:5:90.-   2. An immediate release pharmaceutical formulation as described in    aspect 1 wherein the active ingredient is:-   Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(OMe);-   Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(OMe);-   Ph(3-Cl)(5-OCH₂CH₂F)—(R)CH(OH)C(O)—(S)Aze-Pab(OMe);-   Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab;-   Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(OH);-   Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF);-   Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(OH);-   Ph(3-Cl)(5-OCH₂CH₂F)—(R)CH(OH)C(O)—(S)Aze-Pab; or,-   Ph(3-Cl)(5-OCH₂CH₂F)—(R)CH(OH)C(O)—(S)Aze-Pab(OH).-   3. A solid immediate release pharmaceutical formulation as described    in aspect 1 wherein the active ingredient is:-   Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(OMe);-   Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(OMe); or,-   Ph(3-Cl)(5-OCH₂CH₂F)—(R)CH(OH)C(O)—(S)Aze-Pab(OMe),-   or a pharmaceutically acceptable salt thereof.-   4. A solid immediate release pharmaceutical formulation as described    in aspect 1 wherein the active ingredient is    Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(OMe) or a C₁₋₆    alkanesulfonic acid or an optionally substituted arylsulfonic acid    salt thereof.-   5. An injectable immediate release pharmaceutical formulation as    described in aspect 1 wherein the active ingredient is:-   Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab;-   Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF); or-   Ph(3-Cl)(5-OCH₂CH₂F)—(R)CH(OH)C(O)—(S)Aze-Pab.-   6. The use of a formulation as described in aspect 1 as a    medicament.-   7. The use of a formulation as described in aspect 1 in the    manufacture of a medicament for the treatment of a cardiovascular    disorder.-   8. A method of treating a cardiovascular disorder in a patient    suffering from, or at risk of, said disorder, which comprises    administering to the patient a therapeutically effective amount of a    pharmaceutical formulation as described in aspect 1.-   9. A process for making an immediate release formulation as    described in aspect 1.-   10. The compound    Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(OH).

Also provided is a formulation obtainable by any of the Methods and/orExamples described herein.

1. An immediate release pharmaceutical formulation comprising, as anactive ingredient, a compound of formula (I):

wherein R¹ is C₁₋₂ alkyl substituted with one or more fluorosubstituents; R² is hydrogen, hydroxy, methoxy or ethoxy; and n is 0 or2; or a pharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable diluent or carrier; provided that when the active ingredientis other than in the form of a salt, the formulation does not solelycontain: a solution of one active ingredient and water; a solution ofone active ingredient and dimethylsulphoxide; or a solution of oneactive ingredient in a mixture of ethanol:PEG 660 12-hydroxystearate:water 5:5:90.
 2. An immediate release pharmaceuticalformulation as claimed in claim 1, comprising an acid addition salt of acompound of formula (I) and a pharmaceutically acceptable diluent orcarrier.
 3. An immediate release pharmaceutical formulation as claimedin claim 1, wherein the active ingredient is:Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(OMe);Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(OMe);Ph(3-Cl)(5-OCH₂CH₂F)—(R)CH(OH)C(O)—(S)Aze-Pab(OMe);Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab;Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(OH);Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF);Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(OH);Ph(3-Cl)(5-OCH₂CH₂F)—(R)CH(OH)C(O)—(S)Aze-Pab; orPh(3-Cl)(5-OCH₂CH₂F)—(R)CH(OH)C(O)—(S)Aze-Pab(OH).
 4. A formulation asclaimed in claim 1, wherein the active ingredient is a crystalline saltof: Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(OMe);Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(OMe); orPh(3-Cl)(5-OCH₂CH₂F)—(R)CH(OH)C(O)—(S)Aze-Pab(OMe).
 5. A formulation asclaimed in claim 1, wherein the active ingredient is an ethanesulfonicacid, n-propanesulfonic acid, benzenesulfonic acid,1,5-naphthalenedisulfonic acid, or n-butanesulfonic acid addition saltof Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(OMe) orPh(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(OMe).
 6. Aformulation as claimed in claim 1, wherein the active ingredient isPh(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(OMe), benzene-sulfonic acidsalt, characterised by an X-ray powder diffraction pattern characterisedby peaks with d-values at 5.9, 4.73, 4.09, and 4.08 Å.
 7. A formulationas claimed in claim 1, wherein the active ingredient isPh(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(OMe),hemi-1,5-naphthalenedisulfonic acid salt, characterised by an X-raypowder diffraction pattern characterised by peaks with d-values at 18.3,9.1, 5.6, 5.5, 4.13, 4.02, 3.86, 3.69, and 3.63 Å.
 8. A formulation asclaimed in claim 1, wherein the composition is selected from a solidimmediate release pharmaceutical formulation, an injectable immediaterelease pharmaceutical formulation, or a liquid immediate release oralpharmaceutical formulation.
 9. A method for treating a patient sufferingfrom, or at risk of developing a cardiovascular disorder, comprisingadministering to the patient a therapeutically effective amount of apharmaceutical formulation of any one of claims 1 to 8.